Protein complex using an immunoglobulin fragment and method for the preparation thereof

ABSTRACT

Disclosed are a protein conjugate with improved in vivo duration and stability and the use thereof. The protein conjugate includes a physiologically active polypeptide, a non-peptide polymer and an immunoglobulin Fc fragment. Since the three components are covalently linked, the protein conjugate has extended in vivo duration and enhanced stability for the physiologically active polypeptide. The protein conjugate maintains the in vivo activity at relatively high levels and remarkably increases the serum half-life for the physiologically active polypeptide, with less risk of inducing undesirable immune responses. Thus, the protein conjugate is useful for developing long-acting formulations of various polypeptide drugs.

This is a continuation application of U.S. Ser. No. 12/757,635 filedApr. 9, 2010, which is a continuation application of U.S. Ser. No.10/535,232 filed on Jun. 19, 2006 (U.S. Pat. No. 7,737,260), which is anational stage application under 35 U.S.C. 371 of PCT/KR2004/002944filed on Nov. 13, 2004, which claims priority from Korean patentapplication 10-2003-0080299 filed on Nov. 13, 2003.

TECHNICAL FIELD

The present invention relates to a protein conjugate comprising aphysiologically active polypeptide, a non-peptide polymer and animmunoglobulin Fc fragment, which are covalently linked and have anextended duration of physiological action compared to the native form.

BACKGROUND ART

Since polypeptides tend to be easily denatured due to their lowstability, degraded by proteolytic enzymes in the blood and easilypassed through the kidney or liver, protein medicaments, includingpolypeptides as pharmaceutically effective components, need to befrequently administered to patients to maintain desired blood levelconcentrations and titers. However, this frequent administration ofprotein medicaments, especially through injection, causes pain forpatients. To solve these problems, many efforts have been made toimprove the serum stability of protein drugs and maintain the drugs inthe blood at high levels for a prolonged period of time, and thusmaximize the pharmaceutical efficacy of the drugs. Pharmaceuticalcompositions with sustained activity, therefore need to increase thestability of the protein drugs and maintain the titers at sufficientlyhigh levels without causing immune responses in patients.

To stabilize proteins and prevent enzymatic degradation and clearance bythe kidneys, a polymer having high solubility, such as polyethyleneglycol (hereinafter, referred to simply as “PEG”), was conventionallyused to chemically modify the surface of a protein drug. By binding tospecific or various regions of a target protein, PEG stabilizes theprotein and prevents hydrolysis, without causing serious side effects(Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991).However, despite its capability to enhance protein stability, this PEGcoupling has problems such as greatly reducing the number titers ofphysiologically active proteins. Further, the yield decreases with theincreasing molecular weight of the PEG due to the reduced reactivity ofthe proteins.

Recently, polymer-protein drug conjugates have been suggested. Forexample, as described in U.S. Pat. No. 5,738,846, a conjugate can beprepared by linking an identical protein drug to both ends of PEG toimprove the activity of the protein drug. Also, as described inInternational Pat. Publication No. WO 92/16221, two different proteindrugs can be linked to both ends of PEG to provide a conjugate havingtwo different activities. The above methods, however, were not verysuccessful in sustaining the activity of protein drugs.

On the other hand, Kinstler et al. reported that a fusion proteinprepared by coupling granulocyte-colony stimulating factor (G-CSF) tohuman albumin showed improved stability (Kinstler et al., PharmaceuticalResearch 12(12): 1883-1888, 1995). In this publication, however, sincethe modified drug, having a G-CSF-PEG-albumin structure, only showed anapproximately four-fold increase in residence time in the body and aslight increase in serum half-life compared to the single administrationof the native G-CSF, it has not been industrialized as an effectivelong-acting formulation for protein drugs.

An alternative method for improving the in vivo stability ofphysiologically active proteins is by linking a gene of physiologicallyactive protein to a gene encoding a protein having high serum stabilityby genetic recombination technology and culturing the cells transfectedwith the recombinant gene to produce a fusion protein. For example, afusion protein can be prepared by conjugating albumin, a protein knownto be the most effective in enhancing protein stability, or its fragmentto a physiologically active protein of interest by genetic recombination(International Pat. Publication Nos. WO 93/15199 and WO 93/15200,European Pat. Publication No. 413,622). A fusion protein ofinterferon-alpha and albumin, developed by the Human Genome ScienceCompany and marketed under the trade name of ‘Albuferon™’, increased thehalf-life from 5 hours to 93 hours in monkeys, but it was known to beproblematic because it decreased the in vivo activity to less than 5% ofunmodified interferon-alpha (Osborn et al., J. Phar. Exp. Ther. 303(2):540-548, 2002).

On the other hand, an immunoglobulin (Ig) is composed largely of tworegions: Fab having an antigen-binding site and Fc having acomplement-binding site. Other attempts were made to fuse a protein drugto an immunoglobulin Fc fragment by genetic recombination. For example,interferon (Korean Pat. Laid-open Publication No. 2003-9464), andinterleukin-4 receptor, interleukin-7 receptor or erythropoietin (EPO)receptor (Korean Pat. Registration No. 249572) were previously expressedin mammals in a form fused to an immunoglobulin Fc fragment.International Pat. Publication No. WO 01/03737 describes a fusionprotein comprising a cytokine or growth factor linked to animmunoglobulin Fc fragment through an oligopeptide linker.

In addition, U.S. Pat. No. 5,116,964 discloses an LHR (lymphocyte cellsurface glycoprotein) or CD4 protein fused to an amino terminus orcarboxyl terminus of an immunoglobulin Fc fragment by geneticrecombination, and U.S. Pat. No. 5,349,053 describes a fusion protein ofIL-2 and an immunoglobulin Fc fragment. Other examples of Fc fusionproteins prepared by genetic recombination include a fusion protein ofinterferon-beta or a derivative thereof and an immunoglobulin Fcfragment (International Pat. Publication No. WO 00/23472), a fusionprotein of IL-5 receptor and an immunoglobulin Fc fragment (U.S. Pat.No. 5,712,121), a fusion protein of interferon-alpha and an Fc fragmentof an immunoglobulin G4 (U.S. Pat. No. 5,723,125), and a fusion proteinof CD4 protein and an Fc fragment of an immunoglobulin G2 (U.S. Pat. No.6,451,313). Also, as described in U.S. Pat. No. 5,605,690, an Fc varianthaving an amino acid alteration especially at a complement-binding siteor receptor-binding site can be fused to TNF receptor by recombinant DNAtechnologies to give a TNFR-IgG1 Fc fusion protein. In this way, methodsof preparing an Fc fusion protein using an immunoglobulin Fc fragmentmodified by genetic recombination are disclosed in U.S. Pat. Nos.6,277,375, 6,410,008 and 6,444,792.

U.S. Pat. No. 6,660,843 discloses a method of producing a conjugatecomprising a target protein fused to an immunoglobulin Fc fragment bymeans of a linker in E. coli by genetic recombination. This methodallows the conjugate to be produced at lower cost than when usingmammalian expression systems and provides the conjugate in anaglycosylated form. However, since the target protein and theimmunoglobulin Fc fragment are produced together in E. coli, if thetarget protein is glycosylated in nature, it is difficult to apply sucha target protein using this method. This method has another problem ofexpressing the conjugate as inclusion bodies, resulting in very highmisfolding rates.

However, such Fc fusion proteins produced by genetic recombination havethe following disadvantages: protein fusion occurs only in a specificregion of an immunoglobulin Fc fragment, which is at an amino- orcarboxyl-terminal end; only homodimeric forms and not monomeric formsare produced; and a fusion could take place only between theglycosylated proteins or between the aglycosylated proteins, and it isimpossible to make a fusion protein composed of a glycosylated proteinand an aglycosylated protein. Further, a new amino acid sequence createdby the fusion may trigger immune responses, and a linker region maybecome susceptible to proteolytic degradation.

On the other hand, with respect to the development of fusion proteinsusing an immunoglobulin Fc fragment, there is no report of a conjugatecomprising a target protein linked to a human-derived native Fc using acrosslinking agent. The preparation of a conjugate using a linker hasthe advantages of facilitating the selection and control linking sitesand orientation of two proteins to be linked together, and allowing theexpression in a monomer, dimer or multimer and the preparation ofhomologous or heterogeneous constructs. The immunoglobulin Fc fragmentcan be produced by recombinant DNA technologies using mammalian cells orE. coli. However, to date, there is no report of a native immunoglobulinFc fragment that is singly mass-produced with high yields in E. coli andapplied to long-acting formulations. Also, to date, there has been noattempt for the production of a conjugate comprising a target proteinlinked to such an E. coli-derived immunoglobulin Fc fragment produced byrecombinant DNA technologies by means of a crosslinking agent.

On the other hand, immunoglobulins have antibody functions, such asantibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC), and sugar moieties present atan Fc fragment of immunoglobulins play important roles in the ADCC andCDC effects (Burton D., Molec. Immun. 22, 161-206, 1985).Immunoglobulins lacking sugar moieties have serum half-lives similar toglycosylated immunoglobulins but 10 to 1000-fold reduced complement andreceptor binding affinities (Waldmann H., Eur. J. Immunol. 23, 403-411,1993; Morrison S., J. Immunol. 143, 2595-2601, 1989).

As described above, a variety of methods have been tried for linking apolymer to a physiologically active protein. Conventional methodsenhance the stability of polypeptides but remarkably reduce the activitythereof, or improve the activity of the polypeptides regardless of thestability. Thus, there is a need of a method capable of achieving bothminimal activity reduction and stability enhancement for a protein drug.

In this regard, leading to the present invention, the intensive andthrough research into the development of a long-acting protein drugformulation capable of achieving both minimal activity reduction andstability enhancement, which are conventionally considered difficult toaccomplish, resulted in the finding that a protein conjugate, preparedby covalent bond an immunoglobulin Fc fragment, a non-peptide polymerand a physiologically active polypeptide, remarkably extends the serumhalf-life of the physiologically active protein and maintains highertiters than known protein drugs.

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a proteinconjugate minimizing the activity reduction of a physiologically activepolypeptide while extending the serum half-life of the polypeptide,while reducing the risk of inducing immune responses, and a method ofpreparing such a protein conjugate.

It is another object of the present invention to provide a long-actingprotein drug formulation comprising the protein conjugate with theextended serum half-life as an effective component.

It is a further object of the present invention to provide a method ofimproving the stability and the duration of physiological action byminimizing the activity reduction of a physiologically activepolypeptide while enhancing the serum half-life of the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the results of chromatography of an immunoglobulin Fcfragment obtained by cleavage of an immunoglobulin with papain;

FIG. 2 shows the results of SDS-PAGE of a purified immunoglobulin Fcfragment (M: molecular size marker, lane 1: IgG, lane 2: Fc);

FIG. 3 shows the results of SDS-PAGE of IFNα-PEG-Fc (A),¹⁷Ser-G-CSF-PEG-Fc (B) and EPO-PEG-Fc (C) conjugates, which aregenerated by a coupling reaction (M: molecular size marker, lane 1: Fc,lane 2: physiologically active protein, lane 3: physiologically activeprotein-PEG-Fc conjugate);

FIG. 4 shows the results of size exclusion chromatography of anIFNα-PEG-Fc conjugate that is purified after a coupling reaction;

FIG. 5 shows the results of MALDI-TOF mass spectrometry of an EPO-PEG-Fcconjugate;

FIGS. 6 a and 6 b show the results of MALDI-TOF mass spectrometry andSDS-PAGE analysis, respectively, of a native immunoglobulin Fc and adeglycosylated immunoglobulin Fc (DG Fc);

FIG. 7 shows the results of MALDI-TOF mass spectrometry of anIFNα-PEG-Fc conjugate and an IFNα-PEG-DG Fc conjugate;

FIGS. 8 a to 8 c show the results of reverse phase HPLC of IFNα-PEG-Fc,IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc derivative conjugates;

FIG. 9 is a graph showing the results of pharmacokinetic analysis of anative IFNα, an IFNα-40K PEG complex, an IFNα-PEG-albumin conjugate andan IFNα-PEG-Fc conjugate;

FIG. 10 is a graph showing the results of pharmacokinetic analysis of anative EPO, a highly glycosylated EPO, an EPO-PEG-Fc conjugate and anEPO-PEG-AG Fc conjugate;

FIG. 11 is a graph showing the results of pharmacokinetic analysis ofIFNα-PEG-Fc, IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc derivativeconjugates;

FIG. 12 is a graph showing the pharmacokinetics of a Fab′, a Fab′-S-40KPEG complex, a Fab′-N-PEG-N-Fc conjugate and a Fab′-S-PEG-N-Fcconjugate;

FIG. 13 is a graph showing the in vivo activities of Fab′, a Fab′-S-40KPEG complex, a Fab′-N-PEG-N-Fc conjugate and a Fab′-S-PEG-N-Fcconjugate;

FIG. 14 is a graph showing the results of comparison of human IgGsubclasses for binding affinity to the C1q complement; and

FIG. 15 is a graph showing the results of comparison of a glycosylatedFc, an enzymatically deglycosylated DG Fc and an interferon-PEG-carrierconjugate where the carrier is AG Fc produced by E. coli for bindingaffinity to the C1q complement.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect for accomplishing the above objects, the present inventionprovides a protein conjugate comprising a physiologically activepolypeptide, a non-peptide polymer having a reactive group at both endsand an immunoglobulin Fc fragment, which are covalently linked.

The term “protein conjugate” or “conjugate”, as used herein, refers tocomprise one or more physiologically active polypeptides, one or morenon-peptide polymers having a reactive group at both ends and one ormore immunoglobulin Fc fragments, wherein the three components arecovalently linked. In addition, to be distinguished from the“conjugate”, a construct comprising only two different moleculesselected from a physiologically active polypeptide, a non-peptidepolymer and an immunoglobulin Fc fragment, wherein the two molecules arecovalently linked together, is designated as a “complex”.

The protein conjugate of the present invention is a variant of a proteindrug made to reduce the physiological activity reduction and to increasethe in vivo duration of the protein drug, which is characterized bylinking an immunoglobulin Fc fragment to the protein drug.

The immunoglobulin Fc fragment is safe for use as a drug carrier becauseit is a biodegradable polypeptide that is metabolized in the body. Also,the immunoglobulin Fc fragment has a relatively low molecular weightcompared to the whole immunoglobulin molecules, thus being advantageousin the preparation, purification and yield of conjugates due to. Sincethe immunoglobulin Fc fragment does not contain the Fab fragment, whoseamino acid sequence differs among antibody subclasses and which thus ishighly non-homogenous, it may greatly increase the homogeneity ofsubstances and be less antigenic.

The term “immunoglobulin Fc fragment”, as used herein, refers to aprotein that contains the heavy-chain constant region 2 (C_(H)2) and theheavy-chain constant region 3 (C_(H)3) of an immunoglobulin, and not thevariable regions of the heavy and light chains, the heavy-chain constantregion 1 (C_(H)1) and the light-chain constant region 1 (C_(L)1) of theimmunoglobulin. It may further include the hinge region at theheavy-chain constant region. Also, the immunoglobulin Fc fragment of thepresent invention may contain a portion or all of the heavy-chainconstant region 1 (C_(H)1) and/or the light-chain constant region 1(C_(L)1), except for the variable regions of the heavy and light chains.Also as long as it has a physiological function substantially similar toor better than the native protein the IgG Fc fragment may be a fragmenthaving a deletion in a relatively long portion of the amino acidsequence of C_(H)2 and/or C_(H)3. That is, the immunoglobulin Fcfragment of the present invention may comprise 1) a C_(H)1 domain, aC_(H)2 domain, a C_(H)3 domain and a C_(H)4 domain, 2) a C_(H)1 domainand a C_(H)2 domain, 3) a C_(H)1 domain and a C_(H)3 domain, 4) a C_(H)2domain and a C_(H)3 domain, 5) a combination of one or more domains andan immunoglobulin hinge region (or a portion of the hinge region), and6) a dimer of each domain of the heavy-chain constant regions and thelight-chain constant region.

The immunoglobulin Fc fragment of the present invention includes anative amino acid sequence and sequence derivatives (mutants) thereof.An amino acid sequence derivative is a sequence that is different fromthe native amino acid sequence due to a deletion, an insertion, anon-conservative or conservative substitution or combinations thereof ofone or more amino acid residues. For example, in an IgG Fc, amino acidresidues known to be important in binding, at positions 214 to 238, 297to 299, 318 to 322, or 327 to 331, may be used as a suitable target formodification. Also, other various derivatives are possible, includingone in which a region capable of forming a disulfide bond is deleted, orcertain amino acid residues are eliminated at the N-terminal end of anative Fc form or a methionine residue is added thereto. Further, toremove effector functions, a deletion may occur in a complement-bindingsite, such as a C1q-binding site and an ADCC site. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc fragmentare disclosed in International Pat. Publication Nos. WO 97/34631 and WO96/32478.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of the proteins, or peptides are known in the art (H.Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in bothdirections.

In addition, the Fc fragment, if desired, may be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

The aforementioned Fc derivatives are derivatives that have a biologicalactivity identical to the Fc fragment of the present invention orimproved structural stability, for example, against heat, pH, or thelike.

In addition, these Fc fragments may be obtained from native formsisolated from humans and other animals including cows, goats, swine,mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants orderivatives thereof, obtained from transformed animal cells ormicroorganisms. Herein, they may be obtained from a nativeimmunoglobulin by isolating whole immunoglobulins from human or animalorganisms and treating them with a proteolytic enzyme. Papain digeststhe native immunoglobulin into Fab and Fc fragments, and pepsintreatment results in the production of pF′c and F(ab′)2 fragments. Thesefragments may be subjected, for example, to size exclusionchromatography to isolate Fc or pF′c.

Preferably, a human-derived Fc fragment is a recombinant immunoglobulinFc fragment that is obtained from a microorganism.

In addition, the immunoglobulin Fc fragment of the present invention maybe in the form of having native sugar chains, increased sugar chainscompared to a native form or decreased sugar chains compared to thenative form, or may be in a deglycosylated form. The increase, decreaseor removal of the immunoglobulin Fc sugar chains may be achieved bymethods common in the art, such as a chemical method, an enzymaticmethod and a genetic engineering method using a microorganism. Theremoval of sugar chains from an Fc fragment results in a sharp decreasein binding affinity to the C1q part of the first complement component C1and a decrease or loss in antibody-dependent cell-mediated cytotoxicity(ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducingunnecessary immune responses in vivo. In this regard, an immunoglobulinFc fragment in a deglycosylated or aglycosylated form may be moresuitable to the object of the present invention as a drug carrier.

As used herein, the term “deglycosylation” refers to enzymaticallyremove sugar moieties from an Fc fragment, and the term “aglycosylation”means that an Fc fragment is produced in an unglycosylated form by aprokaryote, preferably E. coli.

On the other hand, the immunoglobulin Fc fragment may be derived fromhumans or other animals including cows, goats, swine, mice, rabbits,hamsters, rats and guinea pigs, and preferably humans. In addition, theimmunoglobulin Fc fragment may be an Fc fragment that is derived fromIgG, IgA, IgD, IgE and IgM, or that is made by combinations thereof orhybrids thereof. Preferably, it is derived from IgG or IgM, which isamong the most abundant proteins in human blood, and most preferablyfrom IgG, which is known to enhance the half-lives of ligand-bindingproteins.

On the other hand, the term “combination”, as used herein, means thatpolypeptides encoding single-chain immunoglobulin Fc regions of the sameorigin are linked to a single-chain polypeptide of a different origin toform a dimer or multimer. That is, a dimer or multimer may be formedfrom two or more fragments selected from the group consisting of IgG1Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc fragments.

The term “hybrid”, as used herein, means that sequences encoding two ormore immunoglobulin Fc fragments of different origin are present in asingle-chain immunoglobulin Fc fragment. In the present invention,various types of hybrids are possible. That is, domain hybrids may becomposed of one to four domains selected from the group consisting ofCH1, CH2, CH3 and CH4 of IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc, and mayinclude the hinge region.

On the other hand, IgG is divided into IgG1, IgG2, IgG3 and IgG4subclasses, and the present invention includes combinations and hybridsthereof. Preferred are IgG2 and IgG4 subclasses, and most preferred isthe Fc fragment of IgG4 rarely having effector functions such as CDC(complement dependent cytotoxicity) (see, FIGS. 14 and 15).

That is, as the drug carrier of the present invention, the mostpreferable immunoglobulin Fc fragment is a human IgG4-derivednon-glycosylated Fc fragment. The human-derived Fc fragment is morepreferable than a non-human derived Fc fragment, which may act as anantigen in the human body and cause undesirable immune responses such asthe production of a new antibody against the antigen.

The present invention is characterized in that the immunoglobulin Fcfragment and the protein drug are linked together via a non-peptidepolymer.

The term “non-peptide polymer”, as used herein, refers to abiocompatible polymer including two or more repeating units linked toeach other by a covalent bond excluding the peptide bond.

The non-peptide polymer capable of being used in the present inventionmay be selected form the group consisting of polyethylene glycol,polypropylene glycol, copolymers of ethylene glycol and propyleneglycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides,dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA(poly(lactic acid) and PLGA (polylactic-glycolic acid), lipid polymers,chitins, hyaluronic acid, and combinations thereof. Most preferred ispoly(ethylene glycol) (PEG). Also, derivatives thereof well known in theart and being easily prepared within the skill of the art are includedin the scope of the present invention. The non-peptide polymerpreferably ranges from 1 to 100 kDa, and preferably 1 to 20 kDa, inmolecular weight. Also, the non-peptide polymer of the presentinvention, linked to the immunoglobulin Fc fragment, may be one polymeror a combination of different types of polymers.

The non-peptide polymer useful in the present invention has a reactivegroup capable of binding to the immunoglobulin Fc fragment and theprotein drug.

The non-peptide polymer has a reactive group at both ends, which ispreferably selected from the group consisting of a reactive aldehydegroup, a propione aldehyde group, a butyl aldehyde group, a maleimidegroup and a succinimide derivative. The succinimide derivative may besuccinimidyl propionate, hydroxy succinimidyl, succinimidylcarboxymethyl or succinimidyl carbonate. In particular, when thenon-peptide polymer has a reactive aldehyde group at both ends, it iseffective in linking at both ends with a physiologically activepolypeptide and an immunoglobulin Fc fragment with minimal non-specificreactions. A final product generated by reductive alkylation via analdehyde bond is much more stable than when linked via an amide bond.

The reactive groups at both ends of the non-peptide polymer may be thesame or different. For example, the non-peptide polymer may possess amaleimide group at one end and, at the other end, an aldehyde group, apropionic aldehyde group or a butyl aldehyde group. When a polyethyleneglycol (PEG) having a reactive hydroxy group at both ends thereof isused as the non-peptide polymer, the hydroxy group may be activated tovarious reactive groups by known chemical reactions, or a PEG having acommercially-available modified reactive group may be used so as toprepare the protein conjugate of the present invention.

On the other hand, in the present invention, a complex of theimmunoglobulin Fc fragment and the non-peptide polymer is linked to aphysiologically active polypeptide to provide a protein conjugate.

The terms “physiologically active polypeptide”, “physiologically activeprotein”, “active polypeptide”, “polypeptide drug” or “protein drug”, asused herein, are interchangeable in their meanings, and are featured inthat they are in a physiologically active form exhibiting various invivo physiological functions.

The protein drug has a disadvantage of being unable to sustainphysiological action for a long period of time due to its property ofbeing easily denatured or degraded by proteolytic enzymes present in thebody. However, when the polypeptide drug is conjugated to theimmunoglobulin Fc fragment of the present invention to form a conjugate,the drug has increased structural stability and degradation half-life.Also, the polypeptide conjugated to the Fc fragment has a much smallerdecrease in physiological activity than other known polypeptide drugformulations. Therefore, compared to the in vivo bioavailability ofconventional polypeptide drugs, the conjugate of the polypeptide and theimmunoglobulin Fc fragment according to the present invention ischaracterized by having markedly improved in vivo bioavailability. Thisis also clearly described through embodiments of the present invention.That is, when linked to the immunoglobulin Fc fragment of the presentinvention, IFNα, G-CSF, hGH and other protein drugs displayed an abouttwo- to six-fold increase in vivo bioavailability compared to theirconventional forms conjugated to PEG alone or both PEG and albumin(Tables 8, 9 and 10).

On the other hand, the linkage of a protein and the immunoglobulin Fcfragment of the present invention is featured in that it is not a fusionby a conventional recombination method. A fusion form of theimmunoglobulin Fc fragment and an active polypeptide used as a drug by arecombination method is obtained in such a way that the polypeptide islinked to the N-terminus or C-terminus of the Fc fragment, and is thusexpressed and folded as a single polypeptide from a nucleotide sequenceencoding the fusion form.

This brings about a sharp decrease in the activity of the resultingfusion protein because the activity of a protein as a physiologicallyfunctional substance is determined by the conformation of the protein.Thus, when a polypeptide drug is fused with Fc by a recombinationmethod, there is no effect with regard to in vivo bioavailability evenwhen the fusion protein has increased structural stability. Also, sincesuch a fusion protein is often misfolded and thus expressed as inclusionbodies, the fusion method is uneconomical in protein production andisolation yield. Further, when the active form of a polypeptide is in aglycosylated form, the polypeptide should be expressed in eukaryoticcells. In this case, Fc is also glycosylated, and this glycosylation maycause unsuitable immune responses in vivo.

That is, only the present invention makes it possible to produce aconjugate of a glycosylated active polypeptide and an aglycosylatedimmunoglobulin Fc fragment, and overcomes all of the above problems,including improving protein production yield, because the two componentsof the complex are individually prepared and isolated by the bestsystems.

On the other hand, the physiologically active polypeptide applicable tothe protein conjugate of the present invention is exemplified byhormones, cytokines, interleukins, interleukin binding proteins,enzymes, antibodies, growth factors, transcription regulatory factors,coagulation factors, vaccines, structural proteins, ligand proteins orreceptors, cell surface antigens, receptor antagonists, and derivativesthereof.

In detail, non-limiting examples of the physiologically activepolypeptide include human growth hormone, growth hormone releasinghormone, growth hormone releasing peptide, interferons and interferonreceptors (e.g., interferon-α, -β and -γ, water-soluble type Iinterferon receptor, etc.), colony stimulating factors, interleukins(e.g., interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12,-13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26,-27, -28, -29, -30, etc.) and interleukin receptors (e.g., IL-receptor,IL-4 receptor, etc.), enzymes (e.g., glucocerebrosidase,iduronate-2-sulfatase, alpha-galactosidase-A, alpha-L-iduronidase,butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase,lipase, uricase, platelet-activating factor acetylhydrolase, neutralendopeptidase, myeloperoxidase, etc.), interleukin and cytokine bindingproteins (e.g., IL-18 bp, TNF-binding protein, etc.), macrophageactivating factor, macrophage peptide, B cell factor, T cell factor,protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin,lymphotoxin, tumor necrosis factor, tumor suppressors, metastasis growthfactor, alpha-1 antitrypsin, albumin, alpha-lactalbumin,apolipoprotein-E, erythropoietin, highly glycosylated erythropoietin,angiopoietins, hemoglobin, thrombin, thrombin receptor activatingpeptide, thrombomodulin, factor VII, factor VIIa, factor VIII, factorIX, factor XIII, plasminogen activating factor, fibrin-binding peptide,urokinase, streptokinase, hirudin, protein C, C-reactive protein, renininhibitor, collagenase inhibitor, superoxide dismutase, leptin,platelet-derived growth factor, epithelial growth factor, epidermalgrowth factor, angiostatin, angiotensin, bone growth factor, bonestimulating protein, calcitonin, insulin, atriopeptin, cartilageinducing factor, elcatonin, connective tissue activating factor, tissuefactor pathway inhibitor, follicle stimulating hormone, luteinizinghormone, luteinizing hormone releasing hormone, nerve growth factors(e.g., nerve growth factor, ciliary neurotrophic factor, axogenesisfactor-1, glucagon-like peptides (e.g., GLP-1 etc.), brain-natriureticpeptide, glial derived neurotrophic factor, netrin, neurophil inhibitorfactor, neurotrophic factor, neuturin, etc.), parathyroid hormone,relaxin, secretin, somatomedin, insulin-like growth factor,adrenocortical hormone, glucagon, cholecystokinin, pancreaticpolypeptide, gastrin releasing peptide, corticotropin releasing factor,thyroid stimulating hormone, autotaxin, lactoferrin, myostatin,receptors (e.g., TNFR(P75), TNFR(P55), IL-1 receptor, VEGF receptor, Bcell activating factor receptor, etc.), receptor antagonists (e.g.,IL1-Ra etc.), cell surface antigens (e.g., CD 2, 3, 4, 5, 7, 11a, 11b,18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies,polyclonal antibodies, antibody fragments (e.g., scFv, Fab, Fab′,F(ab′)2 and Fd), and virus derived vaccine antigens.

In particular, preferred as physiologically active polypeptides arethose requiring frequent dosing upon administration to the body fortherapy or prevention of diseases, which include human growth hormone,interferons (interferon-α, -β, -γ, etc.), granulocyte colony stimulatingfactor, erythropoietin (EPO) and antibody fragments. The most preferablepolypeptide drug is interferon-alpha. In addition, certain derivativesare included in the scope of the physiologically active polypeptides ofthe present invention as long as they have function, structure, activityor stability substantially identical to or improved compared over nativeforms of the physiologically active polypeptides.

In the present invention, an antibody fragment may be Fab, Fab′,F(ab′)2, Fd or scFv, which is capable of binding to a specific antigen,and preferably Fab′. The Fab fragments contain the variable domain(V_(L)) and const domain (C_(L)) of the light chain and the variabledomain (V_(H)) and the first constant domain (C_(H)1) of the heavychain. The Fab′ fragments differ from the Fab fragments in terms ofadding several amino acid residues including one or more cysteineresidues from the hinge region to the carboxyl terminus of the C_(H)1domain. The Fd fragments comprise only the V_(H) and C_(H)1 domain, andthe F(ab′)2 fragments are produced as a pair of Fab′ fragments by eitherdisulfide bonding or a chemical reaction. The scFv (single-chain Fv)fragments comprise the V_(L) and V_(H) domains that are linked to eachother by a peptide linker and thus are present in a single polypeptidechain.

On the other hand, when an immunoglobulin Fc fragment and a protein drugare linked together by means of a non-peptide polymer, linking sites ofthe immunoglobulin Fc fragment include one or more free reactive groupsof amino acid residues present at the hinge region or constant region.Preferably, the immunoglobulin Fc constant region and the protein drugare covalently linked at an amino terminal end, an amino group of alysine residue, an amino group of a histidine residue or a free cysteineresidue to a reactive group at respective ends of the non-peptidepolymer.

The protein conjugate of the present invention may include one or moreunit structures of “physiologically active polypeptide-non-peptidepolymer-immunoglobulin Fc fragment”, wherein all of the components arelinearly linked by a covalent bond. Since the non-peptide polymerpossesses a reactive group at both ends thereof, it is connected to thephysiologically active polypeptide and the immunoglobulin Fc fragmentthrough a covalent bond. That is, to a single immunoglobulin Fcfragment, one or more complexes of a non-peptide polymer with aphysiologically active polypeptide may be linked by a covalent bond toprovide a monomer, dimer or multimer of the physiologically activepolypeptide by means of the immunoglobulin Fc fragment, thereby moreeffectively achieving improved in vivo activity and stability.

In the protein conjugate of the present invention, the physiologicallyactive protein may be linked to the immunoglobulin Fc fragment atvarious molar ratios.

In addition, as conventionally known, two different proteins are linkedtogether via an oligopeptide, an amino acid sequence, created at thejunction site, has a risk of inducing immune responses, and linkingsites of the proteins are limited to an N-terminus and C-terminus. Incontrast, since the protein conjugate of the present invention ismediated by a biocompatible non-peptide polymer, it is advantageous interms of having no side effects such as toxicity or immune responseinduction and allowing the preparation of various protein conjugates dueto its diversity of linking sites.

In addition, the conventional method of directly fusing animmunoglobulin Fc fragment to an active protein by genetic recombinationis problematic because it allows the fusion to be made only in aterminal sequence of the immunoglobulin Fc fragment used as a fusionpartner and because it limits the yield of the fusion protein due to itsproduction mode being dependent on animal cell culture. The conventionalmethod has further problems in which the activity of the active proteinmay decrease due to non-native glycosylation, protein folding mustaccurately occur, and the fusion protein may be produced in a homodimerform. In particular, when conjugates are produced in E. coli, insolublemisfolded conjugates are very difficult to remove. In contrast, theprotein conjugate of the present invention may achieve a much longerduration of action and a much higher stability while not causing theseproblems, is preferable with respect to the maintenance of activity of apolypeptide, and allows the preparation of a conjugate comprising aglycosylated therapeutic protein linked to a non-glycosylated Fc.

On the other hand, low molecular weight chemical binders, such ascarbodiimide or glutaraldehyde, have the following problems: they bindsimultaneously to several sites on a protein, leading to denaturation ofthe protein, and non-specifically bind, thus making it difficult tocontrol linking sites or to purify a connected protein. In contrast,since the protein conjugate of the present invention employs anon-peptide polymer, it facilitates the control of linking sites,minimizes non-specific reactions and facilitates protein purification.

The usefulness of the present invention is described in more detailbased on the embodiments of the present invention, as follows. Theprotein conjugate (polypeptide-PEG-Fc) of the present invention,comprising a physiologically active polypeptide and an immunoglobulin Fcfragment, which are linked to each end of PEG, exerts much higherstability than a polypeptide-PEG complex or a polypeptide-PEG-albuminconjugate. Pharmacokinetic analysis revealed that IFNα has a serumhalf-life increased by about 20 times when linked to 40-kDa PEG(IFNα-40K PEG complex) and by about 10 times in an IFNα-PEG-albuminconjugate, compared to the native IFNα. In contrast, an IFNα-PEG-Fcconjugate according to the present invention showed a half-liferemarkably increased by about 50 times (see, Table 3). In addition, thesame result was observed in other target proteins, human growth hormone(hGH), granulocyte colony-stimulating factor (G-CSF) and its derivative(¹⁷S-G-CSF), or erythropoietin (EPO). Protein conjugates according tothe present invention, each of which comprises a target protein linkedto PEG-Fc, displayed increases about 10-fold in mean residence time(MRT) and serum half-life compared to the native forms of the proteinsand the forms conjugated to PEG or PEG-albumin (see, Tables 4 to 7).

In addition, when a PEG-Fc complex is linked to an —SH group near theC-terminus of a Fab′ or the N-terminus of the Fab′, the resultingFab′-PEG-Fc conjugate displayed a 2 to 3-fold longer serum half-lifethan a 40K PEG-Fab′ complex (see, FIG. 12).

Further, when protein conjugates are prepared using deglycosylatedimmunoglobulin Fc (DG Fc), where sugar moieties are removed, andrecombinant aglycosylated immunoglobulin Fc (AG Fc) derivatives, theirplasma half-lives and in vitro activity were maintained similar to theprotein conjugates prepared using the native Fc (see, Table 3 and FIGS.8 and 11).

Therefore, since the protein conjugates of the present invention haveextended serum half-lives and mean residence time (MRT) when applied toa variety of physiologically active polypeptides including human growthhormone, interferon, erythropoietin, colony stimulating factor or itsderivatives, and antibody derivatives, they are useful for developinglong-acting formulations of diverse physiologically active polypeptides.

In another aspect, the present invention provides a method of preparinga protein conjugate with improved in vivo duration and stability,comprising: (a) facilitating a reaction between a non-peptide polymerhaving a reactive group at both ends thereof, a physiologically activepolypeptide and an immunoglobulin Fc fragment to be covalently linked;and (b) isolating a resulting conjugate comprising the physiologicallyactive polypeptide and the immunoglobulin Fc fragment which are linkedcovalently to each end of the non-peptide polymer.

At the step (a), the covalent linkage of the three components occurssequentially or simultaneously. For example, when the physiologicallyactive polypeptide and the immunoglobulin Fc fragment are linked to eachend of the non-peptide polymer, any one of the physiologically activepolypeptide and the immunoglobulin Fc fragment is linked to one end ofthe non-peptide polymer, and the other is then linked to the other endof the non-peptide polymer. This sequential linkage is preferred forminimizing the production of byproducts other than a desired proteinconjugate.

Thus, the step (a) may include (al) covalently linking an immunoglobulinFc fragment or physiologically active polypeptide to one end of anon-peptide polymer; (a2) isolating a complex comprising theimmunoglobulin Fc fragment or the physiologically active polypeptidelinked to the non-peptide polymer from the reaction mixture; and (a3)covalently linking a physiologically active polypeptide orimmunoglobulin Fc fragment to the other end of the non-peptide polymerof the isolated complex to provide a protein conjugate comprising theimmunoglobulin Fc fragment and the physiologically active polypeptide,which are linked to each end of the non-peptide polymer.

At the step (al), the optimal reaction molar ratio of thephysiologically active polypeptide and the non-peptide polymer may rangefrom 1:2.5 to 1:5, and the optimal reaction molar ratio of theimmunoglobulin Fc fragment and the non-peptide polymer may range from1:5 to 1:10.

On the other hand, at the step (a3), the reaction molar ratio of thecomplex obtained at step (a2) to the immunoglobulin Fc fragment orphysiologically active polypeptide may range from 1:0.5 to 1:20, andpreferably 1:1 to 1:3.

If desired, the steps (al) and (a3) may be carried out in the presenceof a reducing agent depending on the type of reactive groups at bothends of the non-peptide polymer participating in reactions at the steps(al) and (a3). Preferred reducing agents may include sodiumcyanoborohydride (NaCNBH₃), sodium borohydride, dimethylamine borate andpyridine borate.

Taking into consideration purities required at the steps (a2) and (b)and molecular weights and charges of products, a suitable proteinisolation method may be selected from methods commonly used for proteinisolation in the art. For example, a variety of known methods includingsize exclusion chromatography and ion exchange chromatography may beapplied. If desired, a combination of a plurality of different methodsmay be used for a high degree of purification.

In a further aspect, the present invention provides a pharmaceuticalcomposition for providing a physiologically active polypeptide havingimproved in vivo duration and stability, comprising the proteinconjugate of the present invention as an effective component along witha pharmaceutically acceptable carrier.

The term “administration”, as used herein, means introduction of apredetermined amount of a substance into a patient by a certain suitablemethod. The conjugate of the present invention may be administered viaany of the common routes, as long as it is able to reach a desiredtissue. A variety of modes of administration are contemplated, includingintraperitoneally, intravenously, intramuscularly, subcutaneously,intradermally, orally, topically, intranasally, intrapulmonarily andintrarectally, but the present invention is not limited to theseexemplified modes of administration. However, since peptides aredigested upon oral administration, active ingredients of a compositionfor oral administration should be coated or formulated for protectionagainst degradation in the stomach. Preferably, the present compositionmay be administered in an injectable form. In addition, thepharmaceutical composition of the present invention may be administeredusing a certain apparatus capable of transporting the active ingredientsinto a target cell.

The pharmaceutical composition comprising the conjugate according to thepresent invention may include a pharmaceutically acceptable carrier. Fororal administration, the pharmaceutically acceptable carrier may includebinders, lubricants, disintegrators, excipients, solubilizers,dispersing agents, stabilizers, suspending agents, coloring agents andperfumes. For injectable preparations, the pharmaceutically acceptablecarrier may include buffering agents, preserving agents, analgesics,solubilizers, isotonic agents and stabilizers. For preparations fortopical administration, the pharmaceutically acceptable carrier mayinclude bases, excipients, lubricants and preserving agents. Thepharmaceutical composition of the present invention may be formulatedinto a variety of dosage forms in combination with the aforementionedpharmaceutically acceptable carriers. For example, for oraladministration, the pharmaceutical composition may be formulated intotablets, troches, capsules, elixirs, suspensions, syrups or wafers. Forinjectable preparations, the pharmaceutical composition may beformulated into a unit dosage form, such as a multidose container or anampule as a single-dose dosage form. The pharmaceutical composition maybe also formulated into solutions, suspensions, tablets, capsules andlong-acting preparations.

On the other hand, examples of carriers, exipients and diluents suitablefor the pharmaceutical formulations include lactose, dextrose, sucrose,sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acaciarubber, alginate, gelatin, calcium phosphate, calcium silicate,cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oils. Inaddition, the pharmaceutical formulations may further include fillers,anti-coagulating agents, lubricants, humectants, perfumes, emulsifiersand antiseptics.

A substantial dosage of a drug in combination with the Fc fragment ofthe present invention as a carrier may be determined by several relatedfactors including the types of diseases to be treated, administrationroutes, the patient's age, gender, weight and severity of the illness,as well as by the types of the drug as an active component. Since thepharmaceutical composition of the present invention has a very longduration of action in vivo, it has an advantage of greatly reducingadministration frequency of pharmaceutical drugs.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

Example 1 Preparation I of IFNα-PEG-Immunoglobulin Fc Fragment Conjugate<Step 1> Preparation of Immunoglobulin Fc Fragment Using Immunoglobulin

An immunoglobulin Fc fragment was prepared as follows. 200 mg of 150-kDaimmunoglobulin G (IgG) (Green Cross, Korea) dissolved in 10 mM phosphatebuffer was treated with 2 mg of a proteolytic enzyme, papain (Sigma) at37° C. for 2 hrs with gentle agitation. After the enzyme reaction, theimmunoglobulin Fc fragment regenerated thus was subjected tochromatography for purification using sequentially a Superdex column, aprotein A column and a cation exchange column. In detail, the reactionsolution was loaded onto a Superdex 200 column (Pharmacia) equilibratedwith 10 mM sodium phosphate buffer (PBS, pH 7.3), and the column waseluted with the same buffer at a flow rate of 1 ml/min. Unreactedimmunoglobulin molecules (IgG) and F(ab′)2, which had a relatively highmolecular weight compared to the immunoglobulin Fc fragment, wereremoved using their property of being eluted earlier than the Ig Fcfragment. Fab fragments having a molecular weight similar to the Ig Fcfragment were eliminated by protein A column chromatography (FIG. 1).The resulting fractions containing the Ig Fc fragment eluted from theSuperdex 200 column were loaded at a flow rate of 5 ml/min onto aprotein A column (Pharmacia) equilibrated with 20 mM phosphate buffer(pH 7.0), and the column was washed with the same buffer to removeproteins unbound to the column. Then, the protein A column was elutedwith 100 mM sodium citrate buffer (pH 3.0) to obtain highly pureimmunoglobulin Fc fragment. The Fc fractions collected from the proteinA column were finally purified using a cation exchange column (polyCAT,PolyLC Company), wherein this column loaded with the Fc fractions waseluted with a linear gradient of 0.15-0.4 M NaCl in 10 mM acetate buffer(pH 4.5), thus providing highly pure Fc fractions. The highly pure Fcfractions were analyzed by 12% SDS-PAGE (lane 2 in FIG. 2).

<Step 2> Preparation of IFNα-PEG Complex

3.4-kDa polyethylene glycol having an aldehyde reactive group at bothends, ALD-PEG-ALD (Shearwater), was mixed with human interferon alpha-2b(hIFNα-2b, MW: 20 kDa) dissolved in 100 mM phosphate buffer in an amountof 5 mg/ml) at an IFNα:PEG molar ratio of 1:1, 1:2.5, 1:5, 1:10 and1:20. To this mixture, a reducing agent, sodium cyanoborohydride(NaCNBH₃, Sigma), was added at a final concentration of 20 mM and wasallowed to react at 4° C. for 3 hrs with gentle agitation to allow PEGto link to the amino terminal end of interferon alpha. To obtain a 1:1complex of PEG and interferon alpha, the reaction mixture was subjectedto size exclusion chromatography using a Superdex^(R) column(Pharmacia). The IFNα-PEG complex was eluted from the column using 10 mMpotassium phosphate buffer (pH 6.0) as an elution buffer, and interferonalpha not linked to PEG, unreacted PEG and dimer byproducts where PEGwas linked to two interferon alpha molecules were removed. The purifiedIFNα-PEG complex was concentrated to 5 mg/ml. Through this experiment,the optimal reaction molar ratio for IFNα to PEG, providing the highestreactivity and generating the smallest amount of byproducts such asdimers, was found to be 1:2.5 to 1:5.

<Step 3> Preparation of IFNα-PEG-Fc Conjugate

To link the IFNα-PEG complex purified in the above step to theN-terminus of an immunoglobulin Fc fragment, the immunoglobulin Fcfragment (about 53 kDa) prepared in the above step 1 was dissolved in 10mM phosphate buffer and mixed with the IFNα-PEG complex at an IFNα-PEGcomplex:Fc molar ratio of 1:1, 1:2, 1:4 and 1:8. After the phosphatebuffer concentration of the reaction solution was adjusted to 100 mM, areducing agent, NaCNBH₃, was added to the reaction solution at a finalconcentration of 20 mM and was allowed to react at 4° C. for 20 hrs withgentle agitation. Through this experiment, the optimal reaction molarratio for IFNα-PEG complex to Fc, providing the highest reactivity andgenerating the fewest byproducts such as dimers, was found to be 1:2.

<Step 4> Isolation and Purification of the IFNα-PEG-Fc Conjugate

After the reaction of the above step 3, the reaction mixture wassubjected to Superdex size exclusion chromatography so as to eliminateunreacted substances and byproducts and purify the IFNα-PEG-Fc proteinconjugate produced. After the reaction mixture was concentrated andloaded onto a Superdex column, 10 mM phosphate buffer (pH 7.3) waspassed through the column at a flow rate of 2.5 ml/min to remove unboundFc and unreacted substances, followed by column elution to collectIFNα-PEG-Fc protein conjugate fractions. Since the collected IFNα-PEG-Fcprotein conjugate fractions contained a small amount of impurities,unreacted Fc and interferon alpha dimers, cation exchange chromatographywas carried out to remove the impurities. The IFNα-PEG-Fc proteinconjugate fractions were loaded onto a PolyCAT LP column (PolyLC)equilibrated with 10 mM sodium acetate (pH 4.5), and the column waseluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodium acetatebuffer (pH 4.5) using 1 M NaCl. Finally, the IFNα-PEG-Fc proteinconjugate was purified using an anion exchange column. The IFNα-PEG-Fcprotein conjugate fractions were loaded onto a PolyWAX LP column(PolyLC) equilibrated with 10 mM Tris-HCl (pH 7.5), and the column wasthen eluted with a linear gradient of 0-0.3 M NaCl in 10 mM Tris-HCl (pH7.5) using 1 M NaCl, thus isolating the IFNα-PEG-Fc protein conjugate ina highly pure form.

Example 2 Preparation II of IFNα-PEG-Fc Protein Conjugate <Step 1>Preparation of Fc-PEG Complex

3.4-kDa polyethylene glycol having an aldehyde reactive group at bothends, ALD-PEG-ALD (Shearwater), was mixed with the immunoglobulin Fcfragment prepared in the step 1 of Example 1 at Fc:PEG molar ratios of1:1, 1:2.5, 1:5, 1:10 and 1:20, wherein the Ig Fc fragment had beendissolved in 100 mM phosphate buffer in an amount of 15 mg/ml. To thismixture, a reducing agent, NaCNBH₃ (Sigma), was added at a finalconcentration of 20 mM and was allowed to react at 4° C. for 3 hrs withgentle agitation. To obtain a 1:1 complex of PEG and Fc, the reactionmixture was subjected to size exclusion chromatography using aSuperdex^(R) column (Pharmacia). The Fc-PEG complex was eluted from thecolumn using 10 mM potassium phosphate buffer (pH 6.0) as an elutionbuffer, and immunoglobulin Fc fragment not linked to PEG, unreacted PEGand dimer byproducts where PEG was linked to two immunoglobulin Fcfragment molecules were removed. The purified Fc-PEG complex wasconcentrated to about 15 mg/ml. Through this experiment, the optimalreaction molar ratio for Fc to PEG, providing the highest reactivity andgenerating the fewest byproducts such as dimers, was found to be 1:3 to1:10.

<Step 2> Formation and Purification of Conjugate of the Fc-PEG Complexand Interferon Alpha

To link the Fc-PEG complex purified in the above step 1 to theN-terminus of IFNα, the Fc-PEG complex was mixed with IFNα dissolved in10 mM phosphate buffer at Fc-PEG complex: IFNα molar ratios of 1:1,1:1.5, 1:3 and 1:6. After the phosphate buffer concentration of thereaction solution was adjusted to 100 mM, a reducing agent, NaCNBH₃, wasadded to the reaction solution at a final concentration of 20 mM and wasallowed to react at 4° C. for 20 hrs with gentle agitation. After thereaction was completed, unreacted substances and byproducts were removedaccording to the same purification method as in the step 4 of Example 1,thus isolating the Fc-PEG-IFNα protein conjugate in a highly pure form.

Example 3 Preparation of hGH-PEG-Fc Conjugate

An hGH-PEG-Fc conjugate was prepared and purified according to the samemethod as in Example 1, except that drug other than interferon alpha,human growth hormone (hGH, MW: 22 kDa) was used and a hGH:PEG molarratio was 1:5.

Example 4 Preparation of G-CSF-PEG-Fc Conjugate

A G-CSF-PEG-Fc conjugate was prepared and purified according to the samemethod as in Example 1, except that drug other than interferon alpha,human granulocyte colony stimulating factor (hG-CSF), was used and anhG-CSF:PEG molar ratio was 1:5.

On the other hand, a ¹⁷S-G-CSF-PEG-Fc protein conjugate was prepared andpurified according to the same method as described above using a G-CSFderivative, ¹⁷S-G-CSF, having a serine substitution at the seventeenthamino acid residue of the native hG-CSF.

Example 5 Preparation of EPO-PEG-Fc Conjugate

An EPO-PEG-Fc conjugate was prepared and purified according to the samemethod as in Example 1, except that drug other than interferon alpha,human erythropoietin (EPO), was used and an EPO:PEG molar ratio was 1:5.

Example 6 Preparation of Protein Conjugate Using PEG Having DifferentReactive Group

An IFNα-PEG-Fc protein conjugate was prepared using PEG having asuccinimidyl propionate (SPA) reactive group at both ends, as follows.3.4-kDa polyethylene glycol, SPA-PEG-SPA (Shearwater), was mixed with 10mg of interferon alpha dissolved in 100 mM phosphate buffer at IFNα:PEGmolar ratios of 1:1, 1:2.5, 1:5, 1:10 and 1:20. The mixture was thenallowed to react at room temperature with gentle agitation for 2 hrs. Toobtain a 1:1 complex of PEG and interferon alpha (IFNα-PEG complex),where PEG was linked selectively to the amino group of a lysine residueof interferon alpha, the reaction mixture was subjected to Superdex sizeexclusion chromatography. The IFNα-PEG complex was eluted from thecolumn using 10 mM potassium phosphate buffer (pH 6.0) as an elutionbuffer, and interferon alpha not linked to PEG, unreacted PEG and dimerbyproducts in which two interferon alpha molecules were linked to bothends of PEG were removed. To link the IFNα-PEG complex to the aminogroup of a lysine residue of immunoglobulin Fc, the purified IFNα-PEGcomplex was concentrated to about 5 mg/ml, and an IFNα-PEG-Fc conjugatewas prepared and purified according to the same methods as in the steps3 and 4 of Example 1. Through this experiment, the optimal reactionmolar ratio for IFNα to PEG, providing the highest reactivity andgenerating the fewest byproducts such as dimers, was found to be 1:2.5to 1:5.

On the other hand, another IFNα-PEG-Fc conjugate was prepared accordingto the same methods as described above using PEG) having anN-hydroxysuccinimidyl (NHS) reactive group at both ends, NHS-PEG-NHS(Shearwater), or PEG having a buthyl aldehyde reactive group at bothends, BUA-PEG-BUA (Shearwater).

Example 7 Preparation of Protein Conjugate Using PEG Having DifferentMolecular Weight

An IFNα-10K PEG complex was prepared using 10-kDa polyethylene glycolhaving an aldehyde reactive group at both ends, ALD-PEG-ALD(Shearwater). This complex was prepared and purified according to thesame method as in the step 2 of Example 1. Through this experiment, theoptimal reaction molar ratio for IFNα to 10-kDa PEG, providing thehighest reactivity and generating the fewest byproducts such as dimers,was found to be 1:2.5 to 1:5. The purified IFNα-10K PEG complex wasconcentrated to about 5 mg/ml, and, using this concentrate, an IFNα-10KPEG-Fc conjugate was prepared and purified according to the same methodsas in the steps 3 and 4 of Example 1.

Example 8 Preparation of Fab′-S-PEG-N-Fc Conjugate (—SH Group) <Step 1>Expression and Purification of Fab′

An E. coli transformant, BL21/poDLHF (accession number: KCCM-10511),expressing anti-tumor necrosis factor-alpha Fab′, was grown in 100 ml ofLB medium overnight with agitation, and was inoculated in a 5-Lfermentor (Marubishi) and cultured at 30° C. and 500 rpm and at an airflow rate of 20 vvm. To compensate for the insufficient nutrients forbacterial growth during fermentation, the cultures were supplementedwith glucose and yeast extracts according to the fermented states ofbacteria. When the cultures reached an OD₆₀₀ value of 80-100, aninducer, IPTG, was added to the cultures to induce protein expression.The cultures were further cultured for 40 to 45 hrs until the OD valueat 600 nm increased to 120 to 140. The fermentation fluid thus obtainedwas centrifuged at 20,000×g for 30 min. The supernatant was collected,and the pellet was discarded.

The supernatant was subjected to the following three-step columnchromatography to purify anti-tumor necrosis factor-alpha Fab′. Thesupernatant was loaded onto a HiTrap protein G column (5 ml, Pharmacia)equilibrated with 20 mM phosphate buffer (pH 7.0), and the column waseluted with 100 mM glycine (pH 3.0). The collected Fab′ fractions werethen loaded onto a Superdex 200 column (Pharmacia) equilibrated with 10mM sodium phosphate buffer (PBS, pH 7.3), and this column was elutedwith the same buffer. Finally, the second Fab′ fractions were loadedonto a polyCAT 21×250 column (PolyLC), and this column was eluted with alinear NaCl gradient of 0.15-0.4 M in 10 mM acetate buffer (pH 4.5),thus providing highly pure anti-tumor necrosis factor-alpha Fab′fractions.

<Step 2> Preparation and Purification of Fc-PEG Complex

To link a PEG linker to the N-terminus of an immunoglobulin Fc, theimmunoglobulin Fc prepared according to the same method as in the step 1of Example 1 was dissolved in 100 mM phosphate buffer (pH 6.0) at aconcentration of 5 mg/ml, and was mixed with NHS-PEG-MAL (3.4 kDa,Shearwater) at an Fc:PEG molar ratio of 1:10, followed by incubation at4° C. for 12 hrs with gentle agitation.

After the reaction was completed, the reaction buffer was exchanged with20 mM sodium phosphate buffer (pH 6.0) to remove unbound NHS-PEG-MAL.Then, the reaction mixture was loaded onto a polyCAT column (PolyLC).The column was eluted with a linear NaCl gradient of 0.15-0.5 M in 20 mMsodium phosphate buffer (pH 6.0). During this elution, theimmunoglobulin Fc-PEG complex was eluted earlier than unreactedimmunoglobulin Fc, and the unreacted Ig Fc was eluted later, therebyeliminating the unreacted Ig Fc molecules.

<Step 3> Preparation and Purification of Fab′-S-PEG-N-Fc Conjugate (—SHGroup)

To link the immunoglobulin Fc-PEG complex to a cysteine group of theFab′, the Fab′ purified in the above step 1 was dissolved in 100 mMsodium phosphate buffer (pH 7.3) at a concentration of 2 mg/ml, and wasmixed with the immunoglobulin Fc-PEG complex prepared in the above step2 at a Fab′:complex molar ratio of 1:5. The reaction mixture wasconcentrated to a final protein concentration of 50 mg/ml and incubatedat 4° C. for 24 hrs with gentle agitation.

After the reaction was completed, the reaction mixture was loaded onto aSuperdex 200 column (Pharmacia) equilibrated with 10 mM sodium phosphatebuffer (pH 7.3), and the column was eluted with the same buffer at aflow rate of 1 ml/min. The coupled Fab′-S-PEG-N-Fc conjugate was elutedrelatively earlier due to its high molecular weight, and unreactedimmunoglobulin Fc-PEG complex and Fab′ were eluted later, therebyeliminating the unreacted molecules. To completely eliminate unreactedimmunoglobulin Fc-PEG, the collected Fab′-S-PEG-N-Fc conjugate fractionswere again loaded onto a polyCAT 21×250 column (PolyLC), and this columnwas eluted with a linear NaCl gradient of 0.15-0.5 M in 20 mM sodiumphosphate buffer (pH 6.0), thus providing a pure Fab′-S-PEG-N-Fcconjugate comprising the Fc-PEG complex linked to an —SH group near theC-terminus of the Fab′.

Example 9 Preparation of Fab′-N-PEG-N-Fc Conjugate (N-Terminus) <Step 1>Preparation and Purification of Fab′-PEG Complex (N-Terminus)

40 mg of the Fab′ purified in the step 1 of Example 8 was dissolved in100 mM sodium phosphate buffer (pH 6.0) at a concentration of 5 mg/ml,and was mixed with butyl ALD-PEG-butyl ALD (3.4 kDa, Nektar) at aFab′:PEG molar ratio of 1:5. A reducing agent, NaCNBH₃, was added to thereaction mixture at a final concentration of 20 mM, and the reactionmixture was then allowed to react at 4° C. for 2 hrs with gentleagitation.

After the reaction was completed, the reaction buffer was exchanged with20 mM sodium phosphate buffer (pH 6.0). Then, the reaction mixture wasloaded onto a polyCAT column (PolyLC). The column was eluted with alinear NaCl gradient of 0.15-0.4 M in 20 mM acetate buffer (pH 4.5).During this column elution, the Fab′-PEG complex comprising the PEGlinker lined to the N-terminus of the Fab′ was eluted earlier thanunreacted Fab′, and the unreacted Fab′ was eluted later, therebyeliminating the unreacted Fab′ molecules.

<Step 2> Preparation and Purification of Fab′-N-PEG-N-Fc Conjugate

To link the Fab′-PEG complex purified in the above step 1 to theN-terminus of an immunoglobulin Fc, the Fab′-PEG complex was dissolvedin 100 mM sodium phosphate buffer (pH 6.0) at a concentration of 10mg/ml, and was mixed with the immunoglobulin Fc dissolved in the samebuffer at a Fab′-PEG complex:Fc molar ratio of 1:5. After the reactionmixture was concentrated to a final protein concentration of 50 mg/ml, areducing agent, NaCNBH₃, was added to the reaction mixture at a finalconcentration of 20 mM, and the reaction mixture was then reacted at 4°C. for 24 hrs with gentle agitation.

After the reaction was completed, the reaction mixture was loaded onto aSuperdex 200 column (Pharmacia) equilibrated with 10 mM sodium phosphatebuffer (pH 7.3), and the column was eluted with the same buffer at aflow rate of 1 ml/min. The coupled Fab′-N-PEG-N-Fc conjugate was elutedrelatively earlier due to its high molecular weight, and unreactedimmunoglobulin Fc and Fab′-PEG complex were eluted later, therebyeliminating the unreacted molecules. To completely eliminate theunreacted immunoglobulin Fc molecules, the collected Fab′-N-PEG-N-Fcconjugate fractions were again loaded onto a polyCAT 21×250 column(PolyLC), and this column was eluted with a linear NaCl gradient of0.15-0.5 M in 20 mM sodium phosphate buffer (pH 6.0), thus providing apure Fab′-N-PEG-N-Fc conjugate comprising the immunoglobulin Fc-PEGcomplex linked to the N-terminus of the Fab′.

Example 10 Preparation and Purification of Deglycosylated ImmunoglobulinFc

200 mg of an immunoglobulin Fc prepared according to the same method asin Example 1 was dissolved in 100 mM phosphate buffer (pH 7.5) at aconcentration of 2 mg/ml, and was mixed with 300 U/mg of adeglycosylase, PNGase F (NEB). The reaction mixture was allowed to reactat 37° C. for 24 hrs with gentle agitation. Then, to purify thedeglycosylated immunoglobulin Fc, the reaction mixture was loaded onto aSP Sepharose FF column (Pharmacia), and the column was eluted with alinear NaCl gradient of 0.1-0.6 M in 10 mM acetate buffer (pH 4.5) using1 M NaCl. The native immunoglobulin Fc was eluted earlier, and thedeglycosylated immunoglobulin Fc (DG Fc) was eluted later.

Example 11 Preparation of IFNα-PEG-DG Fc Conjugate

To link the deglycosylated immunoglobulin Fc prepared in Example 10 tothe IFNα-PEG complex purified in the step 2 of Example 1, the IFNα-PEGcomplex was mixed with the DG Fc dissolved in 10 mM phosphate buffer atIFNα-PEG complex: DG Fc molar ratios of 1:1, 1:2, 1:4 and 1:8. After thephosphate buffer concentration of the reaction solution was adjusted to100 mM, a reducing agent, NaCNBH₃, was added to the reaction solution ata final concentration of 20 mM and was allowed to react at 4° C. for 20hrs with gentle agitation. Through this experiment, the optimal reactionmolar ratio for IFNα-PEG complex to DG Fc, providing the highestreactivity and generating the fewest byproducts such as dimers, wasfound to be 1:2.

After the coupling reaction, the reaction mixture was subjected to sizeexclusion chromatography using a SuperdexR column (Pharmacia) so as toeliminate unreacted substances and byproducts and purify the IFNα-PEG-DGFc protein conjugate. After the reaction mixture was loaded onto thecolumn, a phosphate buffer (pH 7.3) was passed through the column at aflow rate of 2.5 ml/min to remove unbound DG Fc and unreactedsubstances, followed by column elution to collect IFNα-PEG-DG Fc proteinconjugate fractions. Since the collected IFNα-PEG-DG Fc proteinconjugate fractions contained a small amount of impurities, unreacted DGFc and IFNα-PEG complex, cation exchange chromatography was carried outto remove the impurities. The IFNα-PEG-DG Fc protein conjugate fractionswere loaded onto a PolyCAT LP column (PolyLC) equilibrated with 10 mMsodium acetate (pH 4.5), and the column was eluted with a lineargradient of 0-0.6 M NaCl in 10 mM sodium acetate buffer (pH 4.5) using 1M NaCl. Finally, the IFNα-PEG-DG Fc protein conjugate was purified usingan anion exchange column. The IFNα-PEG-Fc protein conjugate fractionswere loaded onto a PolyWAX LP column (PolyLC) equilibrated with 10 mMTris-HCl (pH 7.5), and the column was then eluted with a linear gradientof 0-0.3 M NaCl in 10 mM Tris-HCl (pH 7.5) using 1 M NaCl, thusisolating the IFNα-PEG-DG Fc protein conjugate in a highly pure form.

Example 12 Preparation and Purification of Recombinant AglycosylatedImmunoglobulin Fc Derivative <Preparation of IgG4 Fc Derivative 1Expression Vector>

To prepare human immunoglobulin IgG4 heavy chain constant regions, afirst derivative (IgG4 delta-Cys), having a nine amino acid deletion atthe amino terminus of the native hinge region, and a second derivative(IgG4 monomer), lacking the hinge region by a deletion of all of twelveamino acids of the hinge region, were prepared. As an expression vectorcontaining an E. coli secretory sequence, pT14S1SH-4T20V22Q (Korean Pat.No. 38061), developed prior to the present invention by the presentinvention, was used.

To obtain human immunoglobulin IgG4 heavy chain constant regions, RT-PCRwas carried out using RNA isolated from human blood cells as a template,as follows. First, total RNA was isolated from about 6 ml of blood usinga Qiamp RNA blood kit (Qiagen), and gene amplification was performedusing the total RNA as a template and a One-Step RT-PCR kit (Qiagen). Inthis PCR, a pair of synthesized primers represented by SEQ ID Nos. 1 and2 and another pair of synthesized primers represented by SEQ ID Nos. 2and 3 were used. SEQ ID NO. 1 is a nucleotide sequence starting from the10th residue, serine, of 12 amino acid residues, below, of the hingeregion of IgG4 (SEQ ID NO: 7). SEQ ID NO. 3 was designed to have anucleotide sequence encoding a C_(H)2 domain having alanine as a firstamino acid residue. SEQ ID NO. 2 was designed to have a BamHIrecognition site containing a stop codon.

1   2   3   4   5   6   7   8   9   10  11  12 (SEQ ID NO: 7)gag tcc aaa tat ggt ccc cca tgc cca tca tgc cca (SEQ ID NO: 8)Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro

To clone each of the amplified IgG4 constant region fragments into anexpression vector containing an E. coli secretory sequence derivative,the pT14S1SH-4T20V22Q (Korean Pat. No. 38061) developed prior to thepresent invention by the present inventors was used. This expressionvector contains a heat-stable enterotoxin secretory sequence derivativethat has a nucleotide sequence represented by SEQ ID NO. 4. Tofacilitate cloning, a StuI recognition site was inserted into an end ofthe E. coli heat-stable enterotoxin secretory sequence derivative of thepT14S1SH-4T20V22Q plasmid through site-directed mutagenesis using a pairof primers represented by SEQ ID Nos. 5 and 6 to induce mutagenesis tointroduce the StuI site at a nucleotide sequence coding for the lastamino acid residue of the secretory sequence. This insertion of the StuIsite was found to be successful by DNA sequencing. The resultingpT14S1SH-4T20V22Q plasmid containing a StuI site was designated as“pmSTII”. The pmSTII plasmid was treated with StuI and BamHI andsubjected to agarose gel electrophoresis, and a large fragment (4.7 kb),which contained the E. coli heat-stable enterotoxin secretory sequencederivative, was purified. Then, the amplified gene fragments weredigested with BamHI and ligated with the linearized expression vector,thus providing pSTIIdCG4Fc and pSTIIG4Mo.

The final expression vectors were individually transformed into E. coliBL21(DE3), and the resulting transformants were designated as“BL21/pSTIIdCG4Fc (HM10932)” and “BL21/pSTIIdCG4Mo (HM10933)”, whichwere deposited at the Korean Culture Center of Microorganisms (KCCM) onSep. 15, 2004 and assigned accession numbers KCCM-10597 and KCCM-10598,respectively. Thereafter, when the cultures reached an OD₆₀₀ value of80, an inducer, IPTG, was added to the cultures to induce proteinexpression. The cultures were further cultured for 40 to 45 hrs untilthe OD value at 600 nm increased to 100 to 120. The E. coli cellscollected from the fermentation fluids were disrupted, and the resultingcell lysates were subjected to two-step column chromatography to purifythe recombinant immunoglobulin constant region derivatives present inthe cytosol of E. coli.

5 ml of a protein-A affinity column (Pharmacia) was equilibrated withPBS, and the cell lysates were loaded onto the column at a flow rate of5 ml/min. Unbound proteins were washed out with PBS, and bound proteinswere eluted with 100 mM citrate (pH 3.0). The collected fractions weredesalted using a HiPrep 26/10 desalting column (Pharmacia) with 10 mMTris buffer (pH 8.0). Then, secondary anion exchange columnchromatography was carried out using 50 ml of a Q HP 26/10 column(Pharmacia). The primary purified recombinant aglycosylatedimmunoglobulin Fc fractions were loaded onto the Q-Sepharose HP 26/10column, and the column was eluted with a linear gradient of 0-0.2 M NaClin 10 mM Tris buffer (pH 8.0), thus providing a highly pure recombinantaglycosylated immunoglobulin Fc (AG Fc) derivative, IgG4 delta-Cys and ahighly pure IgG4 monomer fraction.

Example 13 Preparation of Conjugate of IFNα-PEG Complex and RecombinantAG Fc Derivative

According to the same methods as in Examples 1 and 11, the IFNα-PEGcomplex was linked to the N terminus of the IgG4 delta-Cys as an AG Fcderivative prepared in Example 12. After the coupling reaction,unreacted substances and byproducts were removed from the reactionmixture, and the thus-produced IFNα-PEG-AG Fc protein conjugate (I) wasprimarily purified using 50 ml of a Q HP 26/10 column (Pharmacia) andfurther purified by a high-pressure liquid chromatographic assay using apolyCAT 21.5×250 column (polyLC), thus purifying the conjugate to a highdegree. The coupling reaction solution was desalted using a HiPrep 26/10desalting column (Pharmacia) with 10 mM Tris buffer (pH 8.0). Then, thereaction solution was then loaded onto 50 ml of a Q HP 26/10 column(Pharmacia) at a flow rate of 8 ml/min, and this column was eluted witha linear NaCl gradient of 0-0.2 M to obtain desired fractions. Thecollected fractions were again loaded onto a polyCAT 21.5×250 columnequilibrated with 10 mM acetate buffer (pH 5.2) at a flow rate of 15ml/min, and this column was eluted with a linear NaCl gradient of0.1-0.3 M, thus providing highly pure fractions. According to the samemethod as described above, another IFNα-PEG-AG Fc protein conjugate (II)was prepared using another AG Fc derivative prepared in Example 12, IgG4monomer.

Example 14 Preparation of EPO-PEG-Recombinant AG Fc Derivative Conjugate

According to the same method as in Example 13, an EPO-PEG-recombinant AGFc derivative conjugate was prepared by linking an AG Fc derivative,IgG4 delta-Cys, to the EPO-PEG complex.

Comparative Example 1 Preparation of IFNα-40K PEG Complex

5 mg of interferon alpha was dissolved in 100 mM phosphate buffer toobtain a final volume of 5 ml, and was mixed with 40-kDa activatedmethoxy-PEG-aldehyde (Shearwater), at an IFNα:40-kDa PEG molar ratio of1:4. To this mixture, a reducing agent, NaCNBH₃ was added at a finalconcentration of 20 mM and was allowed to react at 4° C. for 18 hrs withgentle agitation. To inactivate PEG, which did not react with IFNα,Ethanolamine was added to the reaction mixture at a final concentrationof 50 mM.

A Sephadex G-25 column (Pharmacia) was used to remove unreacted PEG andexchange the buffer with another buffer. First, this column wasequilibrated with two column volumes (CV) of 10 mM Tris-HCl buffer (pH7.5), and was loaded with the reaction mixture. Flow throughs weredetected by measuring the absorbance at 260 nm using a UVspectrophotometer. When the column was eluted with the same buffer,interferon alpha modified by adding PEG having a higher molecular weightto its N-terminus was eluted earlier, and unreacted PEG was elutedlater, thus allowing isolation of only IFNα-40K PEG.

The following chromatography was carried out to further purify theIFNα-40K PEG complex from the collected fractions. 3 ml of a PolyWAX LPcolumn (PolyLC) was equilibrated with 10 mM Tris-HCl (pH 7.5). Thecollected fractions containing the IFNα-40K PEG complex was loaded ontothe column at a flow rate of 1 ml/min, and the column was washed with 15ml of the equilibrium buffer. Then, the column was eluted with a linearNaCl gradient of 0-100% using 30 ml of 1 M NaCl, thus eluting interferonalpha conjugated to tri-, di- and mono-PEG, sequentially. To furtherpurify the mono-PEG-conjugated interferon alpha, the collected fractionscontaining the mono-PEG-conjugated interferon alpha were subjected tosize exclusion chromatography. The fractions were concentrated andloaded onto a Superdex 200 column (Pharmacia) equilibrated with 10 mMsodium phosphate buffer (pH 7.0), and the column was eluted with thesame buffer at a flow rate of ml/min. The tri- and di-PEG-conjugatedinterferon alpha molecules were removed based on their property of beingeluted earlier than the mono-PEG-conjugated interferon alpha, thusisolating the mono-PEG-conjugated interferon alpha in a highly pureform.

According to the same method as described above, 40-kDa PEG wasconjugated to the N-terminus of human growth hormone, granulocyte colonystimulating factor (G-CSF), and a derivative of G-CSF, thus providinghGH-40K PEG, G-CSF-40K PEG and 40K PEG-¹⁷S-G-CSF derivative complexes.

Comparative Example 2 Preparation of IFNα-PEG-Albumin Conjugate

To link the IFNα-PEG complex purified in the step 2 of Example 1 to theN-terminus of albumin, the IFNα-PEG complex was mixed with human serumalbumin (HSA, about 67 kDa, Green Cross) dissolved in 10 mM phosphatebuffer at an IFNα-PEG complex:albumin molar ratio of 1:1, 1:2, 1:4 and1:8. After the phosphate buffer concentration of the reaction solutionwas adjusted to 100 mM, a reducing agent, NaCNBH₃, was added to thereaction solution at a final concentration of 20 mM and was allowed toreact at 4° C. for 20 hrs with gentle agitation. Through thisexperiment, the optimal reaction molar ratio for IFNα-PEG complex toalbumin, providing the highest reactivity and generating the fewestbyproducts such as dimers, was found to be 1:2.

After the coupling reaction, the reaction mixture was subjected to sizeexclusion chromatography using a SuperdexR column (Pharmacia) so as toeliminate unreacted substances and byproducts and purify theIFNα-PEG-albumin protein conjugate produced. After the reaction mixturewas concentrated and loaded onto the column, 10 mM sodium acetate bufferpassed through the column at a flow rate of 2.5 ml/min to remove unboundalbumin and unreacted substances, followed by column elution to purifyonly IFNα-PEG-albumin protein conjugate. Since the collectedIFNα-PEG-albumin protein conjugate fractions contained a small amount ofimpurities, unreacted albumin and interferon alpha dimers, cationexchange chromatography was carried out to remove the impurities. TheIFNα-PEG-albumin protein conjugate fractions were loaded onto a SP5PWcolumn (Waters) equilibrated with 10 mM sodium acetate (pH 4.5), and thecolumn was eluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodiumacetate buffer (pH 4.5) using 1 M NaCl, thus isolating theIFNα-PEG-albumin protein conjugate in a highly pure form.

According to the same method as described above, albumin was conjugatedto human growth hormone, G-CSF, and a derivative of G-CSF, thusproviding hGH-PEG-albumin, G-CSF-PEG-albumin and ¹⁷S-G-CSF-PEG-albuminconjugates.

Comparative Example 3 Preparation of Fab′-S-40K PEG Complex

The free cysteine residue of the Fab′ purified in the step 1 of Example8 was activated by incubation in an activation buffer (20 mM sodiumacetate (pH 4.0), 0.2 mM DTT) for 1 hr. After the buffer was exchangedby a PEG modification buffer, 50 mM potassium phosphate (pH 6.5),maleimide-PEG (MW: 40 kDa, Shearwater) was added thereto at aFab′:40-kDa PEG molar ratio of 1:10 and was reacted to react at 4° C.for 24 hrs with gentle agitation.

After the reaction was completed, the reaction solution was loaded ontoa Superdex 200 column (Pharmacia) equilibrated with 10 mM sodiumphosphate buffer (pH 7.3), and the column was eluted with the samebuffer at a flow rate of 1 ml/min. The Fab′ conjugated 40-kDa PEG(Fab′-40K PEG) was eluted relatively earlier due to its high molecularweight, and unreacted Fab′ was eluted later, thereby eliminating theunreacted Fab′. To completely eliminate the unreacted Fab′, thecollected Fab′-40K PEG complex fractions were again loaded onto apolyCAT 21×250 column (PolyLC), and this column was eluted with a linearNaCl gradient of 0.15-0.5 M in 20 mM sodium phosphate buffer (pH 4.5),thus providing a pure Fab′-S-40K PEG complex comprising 40-kDa PEGlinked to an —SH group of the Fab′.

Experimental Example 1 Identification and Quantitative Analysis of theProtein Conjugates <1-1> Identification of the Protein Conjugates

The protein conjugates prepared in the above Examples were analyzed bynon-reduced SDS-PAGE using a 4-20% gradient gel and a 12% gel and ELISA(R&D System).

As a result of SDS-PAGE analysis, as shown in FIG. 3, a couplingreaction of a physiological polypeptide, a non-peptide polymer, PEG, andan immunoglobulin Fc fragment resulted in the successful production ofan IFNα-PEG-Fc conjugate (A), a ¹⁷Ser-G-CSF-PEG-Fc conjugate (B) and anEPO-PEG-Fc conjugate (C).

In addition, the DG Fc prepared in Example 10 was analyzed bynon-reduced 12% SDS-PAGE. As shown in FIG. 6 b, a DG Fc band wasdetected at a position, which corresponds to the molecular weight of thenative Fc lacking sugar moieties.

<1-2> Quantitative Analysis of the Protein Conjugates

The protein conjugates prepared in the above Examples were quantified bysize exclusion chromatography using a HiLoad 26/60 Superdex 75 column(Pharmacia) and 10 mM potassium phosphate buffer (pH 6.0) as an elutionbuffer, wherein a peak area of each protein conjugate was compared tothat of a control group. Previously quantitatively analyzed standards,IFNα, hGH, G-CSF, ¹⁷S-G-CSF, EPO and Fc, were individually subjected tosize exclusion chromatography, and a conversion factor between aconcentration and a peak was determined. A predetermined amount of eachprotein conjugate was subjected to the same size exclusionchromatography. By subtracting a peak area corresponding to animmunoglobulin Fc fragment from the thus-obtained peak area, aquantitative value for a physiologically active protein present in eachprotein conjugate was determined. FIG. 4 shows the result of sizeexclusion chromatography of the purified IFNα-PEG-Fc conjugate, whereina single peak was observed. This result indicates that the purifiedprotein conjugate does not contain multimeric impurities such as adimer, a trimer or a higher number of monomers.

When a physiologically active polypeptide conjugated to Fc wasquantitatively analyzed using an antibody specific to thephysiologically active polypeptide, the antibody was prevented frombinding to the polypeptide, resulting in a value lower than an actualvalue calculated by the chromatography. In the case of the IFNα-PEG-Fcconjugate, an ELISA resulted in an ELISA value corresponding to about30% of an actual value.

<1-3> Evaluation of Purity and Mass of the Protein Conjugates

The protein conjugates prepared in the above Examples were subjected tosize exclusion chromatography, and absorbance was measured at 280 nm. Asa result, the IFNα-PEG-Fc, hGH-PEG-Fc, G-CSF-PEG-Fc and¹⁷Ser-G-CSF-PEG-Fc conjugates displayed a single peak at the retentiontime of a 70 to 80-kDa substance.

On the other hand, reverse phase HPLC was carried out to determinepurities of the protein conjugates prepared in Examples 1, 11 and 13,IFNα-PEG-Fc, IFNα-PEG-DG Fc and IFNα-PEG-recombinant AG Fc derivative. Areverse phase column (259 VHP54 column, Vydac) was used. The column waseluted with a 40-100% acetonitrile gradient with 0.5% TFA, and puritieswere analyzed by measuring absorbance at 280 nm. As a result, as shownin FIG. 8, the samples contain no unbound interferon or immunoglobulinFc, and all of the protein conjugates, IFNα-PEG-Fc (A), IFNα-PEG-DG Fc(B) and IFNα-PEG-recombinant AG Fc derivative (C), were found to have apurity greater than 96%.

To determine accurate molecular weights of the purified proteinconjugates, mass for each conjugate was analyzed using a high-throughputMALDI-TOF mass spectrophotometer (Voyager DE-STR, Applied Biosystems).Sinapinic acid was used as a matrix. 0.5 μl of each test sample wascoated onto a sample slide and air-dried, again mixed with the equalvolume of a matrix solution and air-dried, and introduced into an ionsource. Detection was carried out in a positive fashion using a linearmode TOF analyzer. Ions were accelerated with a split extraction sourceoperated with delayed extraction (DE) using a delayed extraction time of750 nsec to 1500 nsec at a total acceleration voltage of about 2.5 kV.

The molecular weights observed by MALDI-TOF mass spectrometry for the Fcprotein conjugates prepared in Examples are given in Table 1, below.FIG. 5 shows the result of MALDI-TOF mass spectrometry of the EPO-PEG-Fcconjugate, and FIG. 7 shows the results of MALDI-TOF mass spectrometryof the IFNα-PEG-Fc and IFNα-PEG-DG Fc conjugates. As a result, theEPO-PEG-Fc protein conjugate was found to have a purity of more than 95%and a molecular weight very close to a theoretical MW. Also, EPO wasfound to couple to the immunoglobulin Fc fragment at a ratio of 1:1.

TABLE 1 Theoretical MW Measured MW (kDa) (kDa) IFNα-PEG-Fc (E.1) 75.475.9 hGH-PEG-Fc (E.3) 78.4 78.6 G-CSF-PEG-Fc (E.4) 75.3 75.9 ¹⁷S-G-CSFderivative-PEG-Fc 75.0 75.9 (E.4) EPO-PEG-Fc (E.5) 91.4 91.0

In addition, when the Fc and DG Fc prepared in Example 10 were examinedfor their molecular weights by MALDI-TOF mass spectrometry, the DG Fcwas found to be 50 kDa, which is about 3-kDa less than native Fc (FIG. 6a). Since the 3-kDa MW corresponds to the theoretical size of sugarmoieties, the results demonstrate that the sugar moieties are completelyremoved.

Table 2, below, shows the results of MALDI-TOF mass spectrometry of theIFNα-PEG-DG Fc conjugate prepared in Example 11 and theIFNα-PEG-recombinant AG Fc derivative conjugates (I and II) prepared inExample 13. The IFNα-PEG-DG Fc conjugate was found to be 3 kDa lighter,and the IFNα-PEG-recombinant AG Fc derivative conjugate (I) to be about3-4 kDa lighter, than the IFNα-PEG-Fc conjugate of 75.9 kDa. TheIFNα-PEG-recombinant AG Fc derivative conjugate (II) coupled to an Fcmonomer showed a molecular weight decreased by 24.5 kDa corresponding tothe molecular weight of the Fc monomer.

TABLE 2 Theoretical MW Measured MW (kDa) (kDa) IFNα-PEG-DG Fc (E.11)72.8 73.0 IFNα-PEG-recombinant AG 72.3 72.2 Fc derivative (I) (E.13)IFNα-PEG-recombinant AG 46.8 46.6 Fc derivative (II) (E.13)

Experimental Example 2 Pharmacokinetic Analysis I

Native forms of physiologically active proteins (controls) and theprotein conjugates prepared in Examples and Comparative Examples, -40KPEG complexes, -PEG-albumin conjugates, -PEG-Fc conjugates, -PEG-DG Fcconjugates and -PEG-recombinant AG Fc derivative conjugates, wereevaluated for serum stability and pharmacokinetic parameters in SD rats(five rats per group). The controls, and the -40K PEG complexes,-PEG-albumin conjugates, -PEG-Fc conjugates, -PEG-DG Fc conjugates and-PEG-recombinant AG Fc derivative conjugates (test groups) wereindividually injected subcutaneously at a dose of 100 μg/kg. After thesubcutaneous injection, blood samples were collected at 0.5, 1, 2, 4, 6,12, 24, 30, 48, 72 and 96 hrs in the control groups, and, in the testgroups, at 1, 6, 12, 24, 30, 48, 72, 96, 120, 240 and 288 hrs. The bloodsamples were collected in tubes with an anticoagulant, heparin, andcentrifuged for 5 min using an Eppendorf high-speed micro centrifugatorto remove blood cells. Serum protein levels were measured by ELISA usingantibodies specific to the physiologically active proteins.

The results of pharmacokinetic analyses of the native forms of IFNα,hGH, G-CSF and EPO, and -40K PEG complexes thereof, -PEG-albuminconjugates thereof, -PEG-Fc conjugates thereof and -PEG-DG Fc conjugatesthereof, are given in Tables 3 to 7, below. In the following tables,T_(max) indicates the time taken to reach the maximal drug serumconcentration, T_(1/2) indicates the serum half-life of a drug, and MRT(mean residence time) indicates the mean time that a drug moleculeresides in the body.

TABLE 3 Pharmacokinetics of interferon alpha IFNα-PEG- IFNα-PEG- IFNα-IFNα- IFNα- recombinant recombinant IFNα- PEG- PEG- PEG-DG AG Fc AG FcNative 40K PEG albumin Fc Fc derivative derivative IFNα (C.E.1) (C.E.2)(E.1) (E.11) (I) (E.13) (II) (E.13) T_(max) (hr) 1.0 30 12 30 48 24 24T_(1/2) (hr) 1.7 35.8 17.1 90.4 71.0 61.2 31.2 MRT (hr) 2.1 71.5 32.5150.1 120.6 111.0 58.8

TABLE 4 Pharmacokinetics of human growth factor hGH-40K hGH-PEG- NativePEG albumin hGH-PEG- hGH (C.E.1) (C.E.2) Fc (E.3) T_(max) (hr) 1.0 12 1212 T_(1/2) (hr) 1.1 7.7 5.9 11.8 MRT (hr) 2.1 18.2 13.0 18.8

TABLE 5 Pharmacokinetics of G-CSF G-CSF- G-CSF-PEG- G-CSF- Native 40KPEG albumin PEG-Fc G-CSF (C.E.1) (C.E.2) (E.4) T_(max) (hr) 2.0 12 12 12T_(1/2) (hr) 2.8 4.8 5.2 6.9 MRT (hr) 5.2 24.5 25.0 32.6

TABLE 6 Pharmacokinetics of ¹⁷S-G-CSF derivative ¹⁷S-G- Native CSF-40K¹⁷S-G-CSF- ¹⁷S-G- ¹⁷S-G-CSF PEG PEG-albumin CSF-PEG- derivative (C.E.1)(C.E.2) Fc (E.4) T_(max) (hr) 2.0 24 24 24 T_(1/2) (hr) 2.9 4.3 6.4 7.0MRT (hr) 5.8 24.4 25.1 33.2

TABLE 7 Pharmacokinetics of EPO Highly EPO- EPO-PEG- Native glycosylatedPEG-Fc recombinant AG Fc EPO EPO (E.5) derivative (E.13) T_(max) (hr)6.0 12 30 48 T_(1/2) (hr) 9.4 18.4 61.5 87.9 MRT (hr) 21.7 26.8 117.6141.6

As shown from the data of Table 3 and the pharmacokinetic graph of FIG.9, the IFNα-PEG-Fc protein conjugate had a serum half-life of 90.4 hrs,which was about 50 times higher than that of native IFNα and about 2.5times higher than that of IFNα-40K PEG having a half-life of 35.8 hrs,prepared in Comparative Example 1. Also, the IFNα-PEG-Fc proteinconjugate of the present invention was found to be superior in serumhalf-life to IFNα-PEG-albumin, which has a half-life of 17.1 hrs.

On the other hand, as shown in Table 3 and FIG. 11, the IFNα-PEG-DG Fcconjugate had a serum half-life of 71.0 hrs, which was almost the sameas the IFNα-PEG-Fc conjugate, indicating that the deglycosylation of Fcdoes not greatly affect the in vivo stability of the IFNα-PEG-DG Fcconjugate. Also, the conjugate prepared using the recombinant AG Fcderivative produced by a recombinant method was found to have an effectidentical to that of the native form-derived DG Fc. However, the serumhalf-life of a complex coupled to an Fc monomer was about half that of acomplex coupled to a normal Fc dimer.

As shown in Table 4, human growth hormone also showed an extended serumhalf-life when conjugated to the IgG Fc fragment according to thepresent invention. That is, compared to the native form (1.1 hrs), thehGH-40K PEG complex and hGH-PEG-albumin conjugate had slightly increasedhalf-lives of 7.7 hrs and 5.9 hrs, respectively, whereas the hGH-PEG-Fcprotein conjugate of the present invention displayed a greatly extendedserum half-life of 11.8 hrs.

As apparent from the pharmacokinetic data of G-CSF and its derivative inTable 5 and 6, the G-CSF-PEG-Fc and ¹⁷S-G-CSF-PEG-Fc conjugatesdisplayed a much longer serum half-life than the -40K PEG complex and-PEG-albumin conjugate. The immunoglobulin Fc fragment was found in theserum to prolong the duration of action of physiologically activeproteins in native forms, as well as in their derivatives havingalterations of certain amino acid residues in similar levels to thenative forms. From these results, it is easily predictable that themethod of the present invention will have a similar effect on otherproteins and their derivatives.

As shown in Table 7 and FIG. 10, the conjugation of the nativeglycosylated EPO to the Fc fragment also resulted in an increase inserum half-life. That is, EPO had a serum half-life of 9.4 hrs in thenative form, and a prolonged serum half-life of 18.4 hrs when highlyglycosylated to improve serum stability. The EPO-PEG-Fc conjugate,comprising EPO coupled to the immunoglobulin Fc fragment according tothe present invention, displayed a markedly prolonged serum half-life of61.5 hrs. Also, when conjugated to the E. coli-derived recombinantaglycosylated (AG) Fc derivative, the half-life of EPO increased to 87.9hrs, indicating that the aglycosylation of the Fc fragment allows thepreparation of a protein conjugate not affecting serum stability of theprotein without antibody functions.

As apparent from the above results, the protein conjugatescovalent-bonded to the immunoglobulin Fc fragment through a non-peptidepolymer according to the present invention displayed serum half-livesincreased several to several tens to that of the native form. Also, whenthe immunoglobulin Fc was aglycosylated by production in E. coli ordeglycosylated by enzyme treatment, its effect of increasing the serumhalf-life of its protein conjugate was maintained at a similar level.

In particular, compared to proteins modified with 40-kDa PEG having thelongest duration of action among PEG molecules for increasing theduration of action of proteins in the serum, the immunoglobulin Fcprotein conjugates had much superior serum stability. In addition,compared to protein conjugates coupled to albumin instead of theimmunoglobulin Fc, the protein conjugates of the present inventiondisplayed excellent serum stability, indicating that the proteinconjugates of the present invention are effective in developinglong-acting forms of protein drugs. These results, that the presentprotein conjugates have excellent effects on serum stability and MRT ina broad range of proteins including colony stimulating factorderivatives by point mutation compared to conventional PEG- oralbumin-conjugated proteins, indicate that the stability andduration-extending effects of the present protein conjugates areapplicable to other physiologically active polypeptides.

On the other hand, when the IFNα-10K PEG-Fc protein conjugate (Example7) prepared using a non-peptide polymer, 10-kDa PEG, was evaluated forits serum half-life according to the same method as described above, itshowed a serum half-life of 48.8 hrs, which was somewhat shorter thanthe serum half-life (79.7 hrs) of a protein conjugate prepared using3.4-kDa PEG.

In addition, the serum half-lives of the protein conjugates decreasewith increasing molecular weight of the non-peptide polymer PEG. Theseresults indicate that the major factor increasing the serum stabilityand duration of the protein conjugates is the conjugated immunoglobulinFc fragment rather than the non-peptide polymer.

Even when the reactive group of PEG was exchanged with a reactive groupother than the aldehyde group, protein conjugates with the PEG showedsimilar patterns in apparent molecular weight and serum half-life tothose coupled to PEG having an aldehyde reactive group.

Experimental Example 3 Pharmacokinetic Analysis II

To determine the serum half-lives of the Fab′-S-PEG-N-Fc andFab′-N-PEG-N-Fc conjugates prepared in Example 8 and 9 and theFab′-S-40K PEG complex prepared in Comparative Example 3, drugpharmacokinetic analysis was carried out according to the same method asin Experimental Example 2 using Fab′ as a control, the conjugates andthe complex. The results are given in FIG. 12.

As shown in FIG. 12, the Fab′-S-PEG-N-Fc and Fab′-N-PEG-N-Fc conjugatesdisplayed a serum half-life prolonged two or three times compared to theFab′ or Fab′-S-40K PEG complex.

Experimental Example 4 Evaluation of Intracellular Activity of theProtein Conjugates <4-1> Comparison of the IFN Protein Conjugates forIntracellular Activity

To compare the intracellular activity of the IFNα protein conjugates,the IFNα-PEG-Fc (Example 1), IFNα-PEG-DG Fc (Example 11),IFNα-PEG-recombinant AG Fc derivative (Example 13), IFNα-40K PEG(Comparative Example 1) and IFNα-PEG-albumin (Comparative Example 2)were evaluated for antiviral activity by a cell culture bioassay usingMadin Darby Bovine Kidney (MDBK) cells (ATCC CCL-22) infected withvesicular stomatitis virus. Nonpegylated interferon alpha-2b, availablefrom the National Institute for Biological Standards and Controls(NIBSC), was used as a standard material.

MDBK cells were cultured in MEM (minimum essential medium, JBI)supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. under5% CO₂ condition. Samples to be analyzed and the standard material werediluted with the culture medium to predetermined concentrations, and100-μ1 aliquots were placed onto each well of a 96-well plate. Thecultured cells were detached, added to the plate containing the samplesin a volume of 100 μl, and cultured for about 1 hr at 37° C. under 5%CO₂ condition. Then, 50 μl of vesicular stomatitis virus (VSV) of5-7×10³ PFU was added to each well of the plate, and the cells werefurther cultured for about 16 to 20 hrs at 37° C. under 5% CO₂conditions. A well that did not contain the sample or standard materialbut contained only the virus was used as a negative control, and a wellthat contained only cells was used as a positive control.

After the culture medium was removed, 100 μl of a neutral red solutionwas added to the plate to stain viable cells, followed by incubation for2 hrs at 37° C. under 5% CO₂ condition. After the supernatants wereremoved, 100 μl of a 1:1 mixture of 100% ethanol and 1% acetic acid wasadded to each well of the plate. After thorough mixing to dissolve allneutral red crystals eluted from stained cells, absorbance was measuredat 540 nm. The negative control was used as a blank, and ED₅₀ values(doses causing 50% cell growth inhibition) were calculated, where thecell growth of the positive control was set at 100%.

TABLE 8 Relative Specific activity (%) Conc. activity for native (ng/ml)(IU/mg) IFNα Native IFNα 100 4.24E+08 100 IFNα-40K PEG 100 2.04E+07 4.8IFNα-PEG-albumin 100 2.21E+07 5.2 IFNα-PEG-Fc 100 1.19E+08 28.1IFNα-PEG-DG Fc 100 1.09E+08 25.7 IFNα-PEG-recombinant 100 9.58E+07 22.6AG Fc derivative

As shown in Table 8, the IFNα-40K PEG decreased in activity to 4.8% ofthe native IFNα. Especially, as the size of the PEG moieties increased,a protein conjugate has improved serum stability but gradually decreasedactivity. Interferon alpha was reported to have in vitro activities of25% when modified with 12-kDa PEG and about 7% when modified with 40-kDaPEG (P. Bailon et al., Bioconjugate Chem. 12: 195-202, 2001). That is,since a protein conjugate has a longer half-life but sharply decreasesin biological activity as the molecular weight of PEG moieties increase,there is a need for the development of a protein conjugate having alonger serum half-life and a stronger activity. In addition, theIFNα-PEG-albumin conjugate displayed a weak activity of about 5.2%compared to the native IFNα. In contrast, the IFNα-PEG-Fc andIFNα-PEG-DG Fc conjugates of the present invention exhibited a markedlyimproved relative activity of 28.1% and 25.7% compared to the nativeIFNα. Also, the conjugation of IFNα to the recombinant AG Fc derivativeresulted in a similar increase in activity. From these results, it isexpected that interferon alpha conjugated to the immunoglobulin Fcfragment has a markedly increased serum half-life and greatly improvedpharmaceutical efficacy in vivo.

<4-2> Comparison of the Human Growth Hormone Protein Conjugates forIntracellular Activity

To compare the intracellular activity of the human growth hormoneprotein conjugates, the hGH-PEG-Fc, hGH-40K PEG and hGH-PEG-albumin werecompared for intracellular activity.

Intracellular activities of the hGH conjugates were measured by an invitro assay using a rat node lymphoma cell line, Nb2 (EuropeanCollection of Cell Cultures (ECACC) #97041101), which develops humangrowth hormone-dependent mitogenesis.

Nb2 cells were cultured in Fisher's medium supplemented with 10% FBS(fetal bovine serum), 0.075% NaCO₃, 0.05 mM 2-mercaptoethanol and 2 mMglutamin, and were further cultured in a similar medium not containing10% FBS for 24 hrs. Then, the cultured cells were counted, and about2×10⁴ cells were aliquotted onto each well of a 96-well plate. ThehGH-PEG-Fc, the hGH-40K PEG, the hGH-PEG-albumin, a standard availablefrom the National Institute for Biological Standards and Controls(NIBSC) as a control, and native human growth hormone (HM-hGH) werediluted and added to each well at various concentrations, followed byincubation for 48 hrs at 37° C. under 5% CO₂ condition. Thereafter, tomeasure cell proliferation activity by determining the cell number ineach well, 25 μl of the Cell Titer 96 Aqueous One Solution Reagent(Promega) was added to each well, and the cells were further culturedfor 4 hrs. Absorbance was measured at 490 nm, and a titer for eachsample was calculated. The results are given in Table 9, below.

TABLE 9 Relative Specific activity (%) Conc. activity* for native(ng/ml) (U/mg) HM-hGH Native hGH 100  2.71E+06 100 hGH (standard 100 2.58E+06 95.2 available from NIBSC) hGH-40K PEG 100 0.206E+06 7.6hGH-PEG-albumin 100 0.141E+06 5.2 hGH-PEG-Fc 100  0.76E+06 28.1 Specificactivity* = 1/ED₅₀ × 10⁶ (ED₅₀: protein amount required for 50% ofmaximum cell growth

As shown in Table 9, also in the case of human growth hormone, theconjugation to 40-kDa PEG (hGH-40K PEG) resulted in a decrease inactivity to about 7.6% of the native form, and the hGH-PEG-albuminconjugate displayed a low in vitro activity that was about 5.2% of thenative hGH. However, the hGH-PEG-Fc conjugate of the present inventionmarkedly increased in relative activity to greater than 28% compared tothe native hGH. From these results, it is expected that human growthhormone linked to the immunoglobulin Fc fragment has a markedlyincreased serum half-life and a greatly improved in vivo pharmaceuticalefficacy. In addition, it is believed that the increased activity of theimmunoglobulin Fc protein conjugates of the present invention is due tothe increased serum stability and preserved binding affinity toreceptors due to the immunoglobulin Fc or due to the space formed by thenon-peptide polymer. These effects are predicted to be applicable toimmunoglobulin Fc protein conjugates coupled to other physiologicallyactive proteins.

<4-3> Comparison of the G-CSF Protein Conjugates for IntracellularActivity

To compare the intracellular activity of the protein conjugates with aG-CSF derivative, the native G-CSF (Filgrastim, Jeil Pharm. Co., Ltd.),¹⁷Ser-G-CSF derivative, 20K PEG-G-CSF (Neulasta), 40K PEG-¹⁷S-G-CSF,¹⁷Ser-G-CSF-PEG-albumin and ¹⁷S-G-CSF-PEG-Fc were compared forintracellular activity.

First, a human myeloid cell line, HL-60 (ATCC CCL-240, promyelocyticleukemia patient/36 yr old Caucasian female), was cultured in RPMI 1640medium supplemented with 10% FBS. The cultured cells were suspended at adensity of about 2.2×10⁵ cells/ml, and DMSO (dimethylsulfoxide, culturegrade, Sigma) was added thereto at a final concentration of 1.25% (v/v).Then, 90 μl of the cell suspension was seeded onto each well of a96-well plate (Corning/low evaporation 96 well plate), thus providing adensity of about 2×10⁴ cells per well, and cultured in an incubator at37° C. with 5% CO₂ for about 72 hrs.

Each sample, whose protein concentration was determined using a G-CSFELISA kit (R&D systems), was diluted with RPMI 1640 to an identicalconcentration of 10 μg/ml, and further diluted two-fold with RPMI 1640nineteen times. The serial two-fold dilutions were individually added toeach well containing HL-60 cells at a volume of 10 μl, so that theconcentration of each sample started at 1 μg/ml. Then, the cells werecultured in an incubator at 37° C. for 72 hrs.

The proliferation of HL-60 cells was assayed using Cell Titer 96™ (Cat.NO. G4100, Promega), and the increased cell number was determined bymeasuring absorbance at 670 nm.

TABLE 10 Relative activity (%) for ED₅₀ (IU/mg) native G-CSF NativeG-CSF 0.30 100 ¹⁷Ser-G-CSF 0.26 115 G-CSF-20K PEG (Neulasta) 1.20 25¹⁷Ser-G-CSF-40K PEG 10.0 <10.0 ¹⁷Ser-G-CSF-PEG-albumin 1.30 23.0¹⁷Ser-G-CSF-PEG-Fc 0.58 51.7

As shown in Table 10, the immunoglobulin Fc protein conjugates coupledto a G-CSF derivative having an amino acid substitution, ¹⁷Ser-G-CSF,also displayed similar effects to native G-CSF-coupled proteinconjugates. The ¹⁷Ser-G-CSF-PEG was previously reported to have arelatively increased serum half-life but a decreased activity comparedto nonpegylated ¹⁷Ser-G-CSF (Korean Pat. Laid-open Publication No.2004-83268). Especially, as the size of the PEG moieties increased, aprotein conjugate had increased serum stability but gradually decreasedactivity. The ¹⁷Ser-G-CSF-40K PEG showed a very low activity of lessthan about 10% compared to the native form. That is, since a proteinconjugate has an extended serum half-life but a sharply decreasedactivity as the molecular weight of PEG moieties increases, there is theneed for the development of a protein conjugate having a long serumhalf-life and strong activity. The ¹⁷Ser-G-CSF-PEG-albumin also showed alow activity of about 23% compared to the native G-CSF. In contrast, the¹⁷Ser-G-CSF-PEG-Fc was greatly improved in relative activity to morethan 51% compared to the native G-CSF. From these results, it isexpected that ¹⁷Ser-G-CSF linked to the immunoglobulin Fc fragment has amarkedly increased serum half-life and a greatly improved pharmaceuticalin vivo efficacy.

<4-4> Cytotoxicity Neutralization Assay for the Fab′ Conjugates

An in vitro activity assay was carried out using the Fab′-S-PEG-N-Fc andFab′-N-PEG-N-Fc conjugates prepared in Example 8 and 9 and theFab′-S-40K PEG complex prepared in Comparative Example 3. Through acytotoxicity assay based on measuring TNFα-mediated cytotoxicity, theFab′ conjugates were evaluated to determine whether they neutralizeTNFα-induced apoptosis in a mouse fibroblast cell line, L929 (ATCCCRL-2148).

The Fab′-S-PEG-N-Fc and Fab′-N-PEG-N-Fc conjugate and the Fab′-S-40K PEGcomplex were serially two-fold diluted, and 100-μl aliquots were placedonto wells of a 96-well plate. rhTNF-α (R&D systems) and actinomycin D(Sigma) used as an RNA synthesis inhibitor were added to each well atfinal concentrations of 10 ng/ml and 1 μg/ml, respectively, incubatedfor 30 min in an incubator at 37° C. with 5% CO₂, and transferred to amicroplate for assay. L929 cells were added to each well at a density of5×10⁴ cells/50 μl medium and cultured for 24 hrs in an incubator at 37°C. with 5% CO₂. After the culture medium was removed, 50 μl of MTT(Sigma) dissolved in PBS at a concentration of 5 mg/ml was added to eachwell, and the cells were further cultured for about 4 hrs in anincubator at 37° C. with 5% CO₂. 150 μl of DMSO was added to each well,and the degree of cytotoxicity neutralization was determined bymeasuring the absorbance at 540 nm. As a control, the Fab′ purified inthe step 1 of Example 8 was used.

As shown in FIG. 13, all of the protein conjugates used in this test hada similar titer to the Fab′. These results indicate that, when a proteinconjugate is prepared by linking an immunoglobulin Fc to a free cysteineresidue near the N-terminus or C-terminus of a Fab′ through PEG, theFab′ exhibits a markedly increased serum half-life and a high in vivoactivity.

<4-5> Complement-Dependent Cytotoxicity (CDC) Assay

To determine whether the derivatives prepared in Examples and proteinscorresponding to the constant regions of immunoglobulins, expressed inthe E. coli transformants and purified, bind to human C1q, an enzymelinked immunosorbent assay (ELISA) was carried out as follows. As testgroups, immunoglobulin constant regions produced by the HM10932 andHM10927 transformants, deposited at the Korean Culture Center ofMicroorganisms (KCCM) on Sep. 15, 2004, and assigned accession numbersKCCM-10597, KCCM-10588, and the derivatives prepared in the aboveExamples were used. As standards, a glycosylated immunoglobulin(IVIG-globulin S, Green Cross PBM) and several commercially availableantibodies used as therapeutic antibodies were used. The test andstandard samples were prepared in 10 mM carbonate buffer (pH 9.6) at aconcentration of 1 μg/ml. The samples were aliquotted into a 96-wellplate (Nunc) in an amount of 200 ng per well, and the plate was coatedovernight at 4° C. Then, each well was washed with PBS-T (137 mM NaCl, 2mM KCl, mM Na₂HPO₄, 2 mM KH₂PO₄, 0.05% Tween 20) three times, blockedwith 250 μl of a blocking buffer (1% bovine serum albumin in PBS-T) atroom temperature for 1 hr, and washed again with the same PBS-T threetimes. The standard and test samples were diluted in PBS-T to apredetermined concentration and added to antibody-coated wells, and theplate was incubated at room temperature for 1 hr and washed with PBS-Tthree times. Thereafter, 2 μg/ml C1q (R&D Systems) was added to theplate and reacted at room temperature for 2 hrs, and the plate waswashed with PBS-T six times. 200 μl of a 1:1000 dilution of a humananti-human C1q antibody-peroxidase conjugate (Biogenesis, USA) in theblocking buffer was added to each well and reacted at room temperaturefor 1 hr. After each well was washed with PBS-T three times, equalvolumes of color reagents A and B (Color A: stabilized peroxide andColor B: stabilized chromogen; DY 999, R&D Systems) were mixed, and 200μl of the mixture was added to each well, followed by incubation for 30min. Then, 50 μl of a reaction termination solution, 2 M sulphuric acid,was added to each well. The plate was read using a microplate reader(Molecular Device). Absorbance of standard and test samples was measuredat 450 nm, and the results are given in FIGS. 14 and 15, respectively.

When immunoglobulin subclasses were compared with each other forcomplement activity in their immunoglobulin Fc fragment, the highestbinding affinity to C1q was found in human immunoglobulin IgG1(Fitzgerald), the next in IgG2 (Fitzgerald) and then IgG4 (Fitzgerald),indicating that there is a difference between subclasses in complementactivity. The IVIG used in this test, which is a pool of IgG subclasses,exhibited a C1q binding affinity almost the same as the purified IgG1because IgG1 amounts to most of the IVIG. Compared to these standards,with respect to changes in binding affinity to C1q by aglycosylation,IgG1 Fc having the strongest complement activity markedly decreased whenaglycosylated. IgG4 Fc, known not to induce complement activation,rarely had binding affinity to C1q, indicating that the IgG4 Fc is usedas an excellent recombinant carrier with no complement activity (FIG.14).

To determine whether the carrier maintains its property of having nobinding affinity to C1q even after being conjugated to a physiologicallyactive peptide, IFN alpha-Fc conjugates were prepared using glycosylatedFc, enzymatically deglycosylated Fc and aglycosylated recombinant Fc ascarriers for IFN alpha and were evaluated for their binding affinity toC1q. A glycosylated Fc-coupled IFN alpha conjugate (IFNα-PEG-Fc:Glycosylated IgG1Fc) maintained a high binding affinity to C1q. Incontrast, when interferon alpha was coupled to an Fc deglycosylatedusing PNGase F and other enzymes, the resulting conjugate(IFNα-PEG-DGFc: Deglycosylated IgG1Fc) displayed a markedly decreasedbinding affinity to C1q, which was similar to that of the E.coli-derived aglycosylated Fc conjugate. In addition, when the IgG1moiety of the aglycosylated IgG1 Fc-coupled interferon alpha conjugate(IFNα-PEG-AGFcG1: Aglycosylated IgG1Fc) was exchanged with the IgG4moiety, the resulting interferon conjugate (IFNα-PEG-FcG4 derivative 1:Aglycosylated IgG4Fc) was found to completely lose its binding affinityto C1q. When the IgG1 Fc moiety was exchanged with the IgG4 Fc monomer,the resulting conjugate (IFNα-PEG-FcG4 derivative 2: AglycosylatedIgG4Fc). These results indicate that such forms of the IgG4 Fc fragmentare useful as excellent carriers not having the effector functions ofantibody fragments (FIG. 15).

INDUSTRIAL APPLICABILITY

As described hereinbefore, the protein conjugate of the presentinvention greatly increases plasma half-lives of polypeptide drugs tolevels higher than any conventional modified proteins. On the otherhand, the protein conjugates overcome the most significant disadvantageof conventional long-acting formulations, decreasing drug titers, thushaving blood circulation time and in vivo activity superior to albumin,previously known to be most effective. In addition, the proteinconjugates have no risk of inducing immune responses. Due to theseadvantages, the protein conjugates are useful for developing long-actingformulations of protein drugs. The long-acting formulations of proteindrugs according to the present invention are capable of reducing thepatient's pain from frequent injections, and of maintaining serumconcentrations of active polypeptides for a prolonged period of time,thus stably providing pharmaceutical efficacy.

Further, the present method of preparing a protein conjugate overcomesdisadvantages of fusion protein production by genetic manipulation,including difficult establishment of expression systems, glycosylationdifferent from a native form, immune response induction and limitedorientation of protein fusion, low yields due to non-specific reactions,and problems of chemical coupling such as toxicity of chemical compoundsused as binders, thereby easily economically providing protein drugswith extended serum half-life and high activity.

1. A protein conjugate comprising a physiologically active polypeptide,a non-peptide polymer and an immunoglobulin Fc fragment, which arecovalently linked to one another.
 2. The protein conjugate according toclaim 1, wherein the non-peptide polymer is covalently linked via areactive group at both ends thereof to the physiologically activepolypeptide and the immunoglobulin Fc fragment.
 3. The protein conjugateaccording to claim 2, wherein one or more complexes of thephysiologically active polypeptide and the non-peptide polymer arecovalently linked to a single molecule of the immunoglobulin Fcfragment.
 4. The protein conjugate according to claim 1, wherein theimmunoglobulin Fc fragment is non-glycosylated.
 5. The protein conjugateaccording to claim 1, wherein the immunoglobulin Fc fragment is composedof one to four domains selected from the group consisting of C_(H1),C_(H2), C_(H3) and C_(H4) domains.
 6. The protein conjugate according toclaim 5, wherein the immunoglobulin Fc fragment further includes a hingeregion.
 7. The protein conjugate according to claim 1, wherein theimmunoglobulin Fc fragment is selected from the group consisting of Fcfragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybridsthereof.
 8. The protein conjugate according to claim 7, wherein theimmunoglobulin Fc fragment is selected from the group consisting of Fcfragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybridsthereof.
 9. The protein conjugate according to claim 8, wherein theimmunoglobulin Fc fragment is an IgG4 Fc fragment.
 10. The proteinconjugate according to claim 9, wherein the immunoglobulin Fc fragmentis a human aglycosylated IgG4 Fc fragment.
 11. The protein conjugateaccording to claim 2, wherein the reactive group of the non-peptidepolymer is selected from the group consisting of an aldehyde group, apropione aldehyde group, a butyl aldehyde group, a maleimide group and asuccinimide derivative.
 12. The protein conjugate according to claim 11,wherein the succinimide derivative is succinimidyl propionate,succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidylcarbonate.
 13. The protein conjugate according to claim 12, wherein thenon-peptide polymer has a reactive aldehyde group as a reactive group atboth ends thereof.
 14. The protein conjugate according to claim 1,wherein the non-peptide polymer is linked at each end thereof to a freereactive group at an amino terminal end, a lysine residue, a histidineresidue or a cysteine residue of the immunoglobulin Fc fragment and thephysiologically active polypeptide.
 15. The protein conjugate accordingto claim 1, wherein the non-peptide polymer is selected from the groupconsisting of polyethylene glycol single polymers, polypropylene glycolsingle polymers, ethylene glycol-propylene glycol copolymers,polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, dextrans,polyvinyl ethyl ethers, biodegradable polymers, lipid polymers, chitins,hyaluronic acids, and combinations thereof.
 16. The protein conjugateaccording to claim 15, wherein the non-peptide polymer is polyethyleneglycol.
 17. The protein conjugate according to claim 1, wherein thephysiologically active polypeptide is selected from the group consistingof hormones, cytokines, enzymes, antibodies, growth factors,transcription regulatory factors, coagulation factors, vaccines,structural proteins, ligand proteins and receptors.
 18. The proteinconjugate according to claim 17, wherein the physiologically activepolypeptide is selected from the group consisting of human growthhormone, growth hormone releasing hormone, growth hormone releasingpeptide, interferons, interferon receptors, colony stimulating factors,glucagon-like, G-protein-coupled receptor, interleukins, interleukinreceptors, enzymes, interleukin binding proteins, cytokine bindingproteins, macrophage activating factor, macrophage peptide, B cellfactor, T cell factor, protein A, allergy inhibitor, cell necrosisglycoproteins, immunotoxin, lymphotoxin, tumor necrosis factor, tumorsuppressors, metastasis growth factor, alpha-1 antitrypsin, albumin,alpha-lactalbumin, apolipoprotein-E, erythropoietin, highly glycosylatederythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptoractivating peptide, thrombomodulin, factor VII, factor VIIa, factorVIII, factor IX, factor XIII, plasminogen activating factor,fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C,C-reactive protein, renin inhibitor, collagenase inhibitor, superoxidedismutase, leptin, platelet-derived growth factor, epithelial growthfactor, epidermal growth factor, angiostatin, angiotensin, bone growthfactor, bone stimulating protein, calcitonin, insulin, atriopeptin,cartilage inducing factor, elcatonin, connective tissue activatingfactor, tissue factor pathway inhibitor, follicle stimulating hormone,luteinizing hormone, luteinizing hormone releasing hormone, nerve growthfactors, parathyroid hormone, relaxin, secretin, somatomedin,insulin-like growth factor, adrenocortical hormone, glucagon,cholecystokinin, pancreatic polypeptide, gastrin releasing peptide,corticotropin releasing factor, thyroid stimulating hormone, autotaxin,lactoferrin, myostatin, receptors, receptor antagonists, cell surfaceantigens, virus derived vaccine antigens, monoclonal antibodies,polyclonal antibodies, and antibody fragments.
 19. The protein conjugateaccording to claim 18, wherein the physiologically active polypeptide ishuman growth hormone, interferon-alpha, granulocyte colony stimulatingfactor, erythropoietin or a Fab′ antibody fragment.